AU5763600A

AU5763600A - Compositions and methods for assaying subcellular conditions and processes usingenergy transfer

Info

Publication number: AU5763600A
Application number: AU57636/00A
Authority: AU
Inventors: James A. Dykens; Soumitra S. Ghosh; Gonul Velicelebi
Original assignee: Mitokor Inc
Current assignee: Migenix Corp
Priority date: 1999-06-22
Filing date: 2000-06-22
Publication date: 2001-01-09
Also published as: WO2000079274A2; EP1210596A2; JP2003506014A; CA2375542A1; WO2000079274A3

Description

WO 00/79274 PCTUSOO/17380 1 COMPOSITIONS AND METHODS FOR ASSAYING SUBCELLULAR CONDITIONS AND PROCESSES USING ENERGY TRANSFER TECHNICAL FIELD The invention relates generally to biological assays for detecting 5 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 10 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 15 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 20 (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 25 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 WO 00/79274 PCT/USOO/17380 2 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 5 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 10 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. 15 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 20 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 25 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 30 compositions for monitoring and assaying processes within subcellular compartments WO 00/79274 PCT/USOO/17380 3 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 5 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. 10 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) 15 techniques. Such ET-based methods and compositions further provide means to screen for and identify agents that alter (e.g., increase or decrease in a statistically significant manner) 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, 20 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 25 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 WO 00/79274 PCT/USOO/1 7380 4 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 5 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 10 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 15 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 20 indicator of fluid volume. In other embodiments of the invention, the sample comprises one or more mitochondria that are present within at least one cell, and the signal detected is compared with a reference signal. In certain further embodiments the reference signal is generated from a subcellular site that may be a mitochondrial outer membrane, 25 mitochondrial inner membrane, mitochondrial intermembrane space, mitochondrial matrix, cytoplasm, nucleus, nuclear membrane or plasma membrane. In another embodiment the reference signal is generated from extracellular medium. In another embodiment mitochondria are present within at least one cell during at least one step, and in certain further embodiments the cell is an organism, a cultured cell, a cybrid cell, 30 a plant cell or an animal cell. In certain other embodiments the cell is present in a WO 00/79274 PCTIUS0O/17380 5 biological sample derived from a multicellular organism, which in some embodiments is a plant cell and in other embodiments is an animal cell; in some embodiments the animal is a mammal that in some embodiments is a human. In a further embodiment the human has, is suspected of having or is at risk of having a disease or disorder 5 associated with organellar dysfunction, which in certain further embodiments is mitochondrial dysfunction and in certain other embodiments is lysosomal dysfunction. In another embodiment of this aspect of the invention, the first energy transfer molecule localizes to a submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane, and the second energy transfer molecule localizes to a 10 submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane. In one embodiment the concentration of the first energy transfer molecule in the submitochondrial site does not change as a function of membrane potential, and the concentration of the second energy transfer molecule in the mitochondrial matrix decreases as a function of membrane potential. In another embodiment the first energy 15 transfer molecule has an excitation maximum at a wavelength of from about 373 nm to about 390 nm, and an emission maximum at a wavelength of from about 400 nm to about 500 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 400 nm to about 500 nm. In a further embodiment the first energy transfer molecule is a fusion protein, wherein the fusion protein comprises a 20 blue-shifted green fluorescent protein polypeptide having a mutation in at least one of Phe-64, Ser-65, Tyr-66, Val-68 and Tyr-145, and a polypeptide sequence that localizes the fusion protein to a submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane; and the second energy transfer molecule is DASPEI, DASPMI, 4-Di-l-ASP, 2-Di-l-ASP, DiOC 7 (3), DiOC 6 (3), JC-1 or SYTO@ 18 yeast 25 mitochondrial stain. In another embodiment the first energy transfer molecule has an excitation maximum at a wavelength of from about 425 nm to about 440 nm, and an emission maximum at a wavelength of from about 450 nm to about 535 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 450 nm to about 530 inn.

WO 00/79274 PCT/US0O/17380 6 In another embodiment the first energy transfer molecule is a fusion protein, wherein the fusion protein comprises a cyan-shifted Green Fluorescent Protein polypeptide having a mutation in at least one of Phe-64, Ser-65, Tyr-66, Asn-146, Met 153, Val-163 and Asn-212, and a polypeptide sequence that localizes the fusion protein 5 to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane; and the second energy transfer molecule is DASPEI, 2-Di-1-ASP, DiOC 6 (3), SYTO@ 18 yeast mitochondrial stain, rhodamine 6G, JC-1, NBD C6-ceramide or NBD C6-sphingomyelin. In another embodiment the first energy transfer molecule has an excitation maximum at a wavelength of from about 470 10 nm to about 500 nm, and an emission maximum at a wavelength of from about 505 nm to about 565 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 505 nm to about 565 nm. In yet another embodiment, the first energy transfer molecule is nonylacridine orange, MitoTracker® Green FM, MitoFluorTM Green or a fusion protein, 15 wherein the fusion protein comprises a Green Fluorescent Protein polypeptide that is a wildtype Green Fluorescent Protein polypeptide, a red-shifted Green Fluorescent Protein polypeptide having a mutation in one or more of Phe-64, Ser-65, Tyr-66, Gln 69, Ser-72 and Thr-203 or a yellow-shifted Green Fluorescent Protein polypeptide having a mutation in one or more of Phe-64, Ser-65, Tyr-66, Gln-69, Ser-72 and Thr 20 203, and a polypeptide sequence that localizes the fusion protein to a submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane; and the second energy transfer molecule is rhodamine 123, JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylrosamine or rhodamine B. In another embodiment, the first energy transfer molecule has an excitation maximum at a wavelength of from about 25 545 to about 560 nm, and an emission maximum at a wavelength of from about 565 to about 625 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 565 to about 625 nm. In an further embodiment the first energy transfer molecule is MitoTracker@ Orange CMTMRos; and the second energy transfer molecule is DiOC 2 (5). In another embodiment the first energy transfer 30 molecule has an excitation maximum at a wavelength of from about 495 to about 510 WO 00/79274 PCTIUSOO/17380 7 nm, and an emission maximum at a wavelength of from about 510 to about 570 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 510 to about 560 nm. In another embodiment the first energy transfer molecule is a fusion protein, wherein the fusion protein comprises a polypeptide 5 sequence that is a FLASH protein sequence or a yellow-shifted Green Fluorescent Protein polypeptide sequence having a mutation in one or more of Ser-65, Tyr-66, Ser 72 and Thr-203, and a polypeptide sequence that localizes the fusion protein to a submitochondrial site that is mitochondrial matrix and mitochondrial inner membrane; and the second energy transfer molecule is JC-1, tetrabromorhodamine 123, rhodamine 10 6G, TMRM, TMRE, tetramethylrosamine, rhodamine B and 4-dimethylamino tetramethylrosamine. In another embodiment of this aspect of the invention, a relative amount of the signal generated by energy transfer is detected. In certain other embodiments the signal is detected over a period of time and a rate of change in the signal level is 15 determined, and in certain other embodiments the signal is detected over a period of time and integrated. In another embodiment membrane potential comprises an electric potential, a pH potential, or both. In one embodiment the first and second energy transfer molecules localize to within from about 10 angstroms to about 100 angstroms of each other, and in another embodiment they localize to within from about 10 20 angstroms to about 50 angstroms of each other and in another embodiment they localize to within from about 20 angstroms to about 50 angstroms of each other. In certain embodiments the signal is generated by fluorescence resonance energy transfer. Turning to another aspect, the present invention provides a method for identifying an agent that alters mitochondrial membrane potential, comprising the steps 25 of contacting, in the absence and presence of a candidate agent, 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 30 adjacent submitochondrial sites, the sites being mitochondrial outer membrane, WO 00/79274 PCT/USOO/17380 8 mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and 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; detecting a signal 5 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; and comparing the signal generated in the absence of the candidate agent to the signal generated in the presence of the candidate agent, and therefrom identifying an agent that 10 alters mitochondrial membrane potential. In another aspect the invention provides a method for identifying a regulator of an agent that alters mitochondrial membrane potential, comprising the steps of contacting, in the absence and presence of a candidate regulator, an agent that alters mitochondrial membrane potential including such an agent identified according to the 15 method provided hereinabove and 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 20 mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and 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; detecting a signal generated by energy transfer from the first energy 25 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; and comparing the signal generated in the absence of the candidate regulator to the signal generated in the presence of the candidate regulator, and therefrom identifying a regulator of an agent that alters mitochondrial membrane 30 potential. In one embodiment the regulator is an agonist of the agent that alters WO 00/79274 PCTUSOO/17380 9 mitochondrial potential, and in another embodiment the regulator is an antagonist of the agent that alters mitochondrial potential. In another embodiment the agent that alters mitochondrial membrane potential is an apoptogen. In another embodiment the agent that alters mitochondrial membrane potential is thapsigargin, an ionophore or an 5 excitatory amino acid or derivative thereof. In certain further embodiments the ionophore is ionomycin or A23187. In certain other embodiments the excitatory amino acid or derivative thereof is glutamate, NAAG, NMDA, AMPA, APPA or kainate. Turning now to another aspect, the invention provides a method for identifying an agent that preferentially alters mitochondrial membrane potential in 10 mitochondria from a first biological source without substantially altering mitochondrial membrane potential in mitochondria from a second biological source, comprising the steps of contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more mitochondria simultaneously or sequentially and in either order with each of a first and a second energy transfer 15 molecule that is not endogenous to the mitochondria, wherein the first sample is derived from a first biological source and the second sample is derived from a second biological source that is distinct from the first biological source, 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 20 mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and 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 in the presence of each of the first and second samples; detecting a 25 signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule in the presence of each of the first and second samples, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential; and comparing the signal generated in the presence of each of the first and second samples in the absence of the 30 candidate agent to the signal generated in the presence of each of the first and second WO 00/79274 PCT/USOO/17380 10 samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters mitochondrial membrane potential In one embodiment the first and second biological sources are distinct biological species, and in another embodiment the first biological source is a mammal 5 suspected of having, diagnosed as having or predisposed to having a disease, and the second biological source is a mammal that is not suspected of having and has not been diagnosed as having or predisposed to having the disease. In a further embodiment the first biological source is a human and the second biological source is a human. In another embodiment the disease is Alzheimer's disease, Parkinson's disease or type II 10 diabetes. The present invention provides, in another aspect, a method for identifying an agent that preferentially alters mitochondrial membrane potential in mitochondria from a first biological sample without substantially altering mitochondrial membrane potential in mitochondria from a second biological sample, comprising the 15 steps of contacting, in the absence and presence of a candidate agent, each of a first and a second biological 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 sample is derived from a first tissue and the second sample is derived from a second tissue that is distinct 20 from the first tissue, 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 the first energy transfer molecule is an energy donor molecule and the second energy 25 transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule in the presence of each of the first and second samples; detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule in the presence of each of the first and second samples, wherein the concentration of at least one of the energy transfer 30 molecules in the mitochondria changes as a function of membrane potential; and WO 00/79274 PCTUSOO/17380 11 comparing the signal generated in the presence of each of the first and second samples in the absence of the candidate agent to the signal generated in the presence of each of the first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters mitochondrial membrane potential. In one 5 embodiment the first tissue and the second tissues are derived from the same subject, while in another embodiment the first and second tissues are each derived from a subject of the same species. In another embodiment the first and second tissues are derived from subjects of distinct species. It is still another aspect of the invention to provide a method of detecting 10 the fusion of a first mitochondrion and a second mitochondrion. comprising the steps of contacting a first sample comprising one or more mitochondria with a first energy transfer molecule that is not endogenous to the mitochondria; contacting a second sample comprising one or more mitochondria with a second energy transfer molecule that is not endogenous to the mitochondria; wherein the first and second energy transfer 15 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 the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; contacting the first 20 sample with the second sample under conditions and for a time sufficient to permit mitochondrial fusion; 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, and therefrom determining fusion of the first mitochondrion and the second mitochondrion. 25 The invention provides, in another aspect, a method of identifying an agent that alters the fusion of mitochondria, comprising the steps of contacting a first sample comprising one or more mitochondria with a first energy transfer molecule that is not endogenous to the mitochondria; contacting a second sample comprising one or more mitochondria with a second energy transfer molecule that is not endogenous to the 30 mitochondria; wherein the first and second energy transfer molecules each localize WO 00/79274 PCTUSOO/17380 12 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 the first energy transfer molecule is an energy donor molecule and the second energy 5 transfer molecule is an energy acceptor molecule; contacting, in the absence and presence of a candidate agent, the first sample with the second sample under conditions and for a time sufficient to permit mitochondrial fusion; exciting the energy donor molecule to produce an excited energy donor molecule; detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer 10 molecule; and comparing the signal detected in the absence of the candidate agent to the signal detected in the presence of the candidate agent, and therefrom identifying an agent that alters the fusion of the mitochondria. In certain embodiments the agent increases mitochondrial membrane potential, in certain other embodiments the agent dissipates mitochondrial membrane potential, in certain other embodiments the agent 15 collapses mitochondrial membrane potential, and in certain embodiments the agent alters an equilibrium distribution of at least one ionic species on either side of a cellular membrane. In a further embodiment the ionic species is Ca2 and the cellular membrane is a mitochondrial membrane. In certain embodiments the agent that collapses mitochondrial membrane potential is an apoptogen, and in certain other 20 embodiments the agent that collapses mitochondrial membrane potential interacts with an adenine nucleotide translocator, and in certain other embodiments the agent that collapses mitochondrial membrane potential is atractyloside, carboxyatractyloside, bongkrekic acid or isobongkrekic acid. Turning to another aspect, the invention provides a reagent for 25 measuring mitochondrial Ay, comprising a FRET donor molecule and a FRET acceptor molecule, wherein the accumulation of at least one of the molecules in mitochondria is dependent on Ay and the accumulation of the other of the molecules in mitochondria is independent of Ay. In one embodiment the molecule that accumulates in mitochondria independent of Ay is NAO, MitoTracker@ Green FM, MitoFluorTM, DAPI, or a fusion 30 protein comprising a polypeptide that is a red- shifted Green Fluorescent Protein WO 00/79274 PCT/USO0/17380 13 polypeptide, a yellow-shifted Green Fluorescent Protein polypeptide or a "FLASH" polypeptide, and a polypeptide sequence that localizes the fusion protein to the mitochondrial matrix or inner membrane. In certain other embodiments the molecule that accumulates in mitochondria in a manner dependent on Ay is TMRM, TMRE, 5 rhodamine 123, ethidum bromide, 4-Di-1-ASP, 2-Di-1-ASP or DASPEI. The invention also provides, in certain embodiments, a kit comprising the reagent just described and ancillary reagents for measuring mitochondrial Ay. It is another aspect of the present invention to provide a method for assaying cellular membrane potential, comprising the steps of: contacting a sample 10 comprising at least one cellular membrane, simultaneously or sequentially and in either order, with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a 15 cellular membrane that forms a subcellular compartment, and 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 20 concentration of at least one of the energy transfer molecules in the membrane site changes as a function of membrane potential. In one embodiment the first energy transfer molecule localizes to a first membrane site that is mitochondria, endoplasmic reticulum, Golgi, lysosome or plasma membrane and the second energy transfer molecule localizes to the same membrane site or to an acceptably adjacent membrane 25 site that is mitochondria, endoplasmic reticulum, Golgi, lysosome or plasma membrane. In another embodiment the concentration of the first energy transfer molecule in the first membrane site does not change as a function of membrane potential, and the concentration of the second energy transfer molecule in the membrane site decreases as a function of membrane potential.

WO 00/79274 PCT/USOO/17380 14 In one embodiment the first energy transfer molecule has an excitation maximum at a wavelength of from about 373 nm to about 390 nm, and an emission maximum at a wavelength of from about 400 nm to about 500 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 400 5 nm to about 500 nm. In a further embodiment the first energy transfer molecule has an excitation maximum at a wavelength of from about 425 nm to about 440 nm, and an emission maximum at a wavelength of from about 450 nm to about 535 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 450 nm to about 530 nm. In another embodiment the first energy transfer 10 molecule has an excitation maximum at a wavelength of from about 470 nm to about 500 nm, and an emission maximum at a wavelength of from about 505 nm to about 565 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 505 nm to about 565 nm. In another embodiment the first energy transfer molecule has an excitation maximum at a wavelength of from about 545 15 to about 560 nm, and an emission maximum at a wavelength of from about 565 to about 625 nm; and the second energy transfer molecule has an excitation maximum at a wavelength of from about 565 to about 625 nm. In yet another aspect, the invention provides a method for identifying an agent that alters a cellular membrane potential, comprising the steps of contacting, in 20 the absence and presence of a candidate agent, a sample comprising one or more cellular membranes simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such 25 that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and 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; detecting a signal generated by energy transfer from the first energy transfer 30 molecule to the second energy transfer molecule, wherein the concentration of at least WO 00/79274 PCT/USOO/17380 15 one of the energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and comparing the signal generated in the absence of the candidate agent to the signal generated in the presence of the candidate agent, and therefrom identifying an agent that alters cellular membrane potential. 5 Another aspect of the invention is to provide a method for identifying a regulator of an agent that alters cellular membrane potential, comprising the steps of contacting, in the absence and presence of a candidate regulator, an agent that alters a cellular membrane potential (which may be an agent identified according to the method just described) and a sample comprising one or more cellular membranes 10 simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular 15 compartment, and 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; 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 20 transfer molecules in the subcellular compartment changes as a function of membrane potential; and comparing the signal generated in the absence of the candidate regulator to the signal generated in the presence of the candidate regulator, and therefrom identifying a regulator of an agent that alters cellular membrane potential. In another aspect the invention provides a method for identifying an 25 agent that preferentially alters a cellular membrane potential in a membrane from a first biological source without substantially altering cellular membrane potential in a membrane from a second biological source, comprising the steps of contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more cellular membranes simultaneously or sequentially and 30 in either order with each of a first and a second energy transfer molecule that is not WO 00/79274 PCTIUSO0/17380 16 endogenous to the sample, wherein the first sample is derived from a first biological source and the second sample is derived from a second biological source that is distinct from the first biological source, the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably 5 adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and 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 in the presence of each of the first and second samples; 10 detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule in the presence of each of the first and second samples, wherein the concentration of at least one of the energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and comparing the signal generated in the presence of each of the first and second samples 15 in the absence of the candidate agent to the signal generated in the presence of each of the first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters cellular membrane potential Turning to another aspect, the invention provides a method for identifying an agent that preferentially alters a cellular membrane potential in a 20 membrane from a first biological sample without substantially altering a cellular membrane potential in a membrane from a second biological sample, comprising the steps of contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more cellular membranes simultaneously or sequentially and in either order with each of a first and a second energy transfer 25 molecule that is not endogenous to the sample, wherein the first sample is derived from a first tissue and the second sample is derived from a second tissue that is distinct from the first tissue, the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a 30 cellular membrane that forms a subcellular compartment, and the first energy transfer WO 00/79274 PCT/USOO/17380 17 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 in the presence of each of the first and second samples; detecting a signal generated by energy transfer from the first energy transfer molecule to 5 the second energy transfer molecule in the presence of each of the first and second samples, wherein the concentration of at least one of the energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and comparing the signal generated in the presence of each of the first and second samples in the absence of the candidate agent to the signal generated in the presence of each of 10 the first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters a cellular membrane potential. In still another aspect the invention provides a method for detecting a specific type of cell in a sample, comprising the steps of contacting a sample comprising one or more mitochondria simultaneously or sequentially and in either order 15 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 subcellular site or to acceptably adjacent subcellular sites, and the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; exciting the energy 20 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 at least one of the energy transfer molecules preferentially accumulates in the specific type of cell; wherein the signal correlates with the presence of the specific type of cell in the sample. In one embodiment the method 25 further comprises the step of comparing the signal generated in the sample with the signal generated from a control sample lacking the specific type of cell. In another embodiment the specific type of cell is a cancer cell. In another aspect the invention provides a method for identifying a AYm stabilizing agent, comprising the steps of contacting, in the absence and presence of a 30 candidate Aym stabilizing agent, an agent that alters Aym and a sample comprising one WO 00/79274 PCTUSOO/17380 18 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 5 submitochondrial sites that are mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and 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; detecting a signal generated by energy 10 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; and comparing the signal generated in the absence of the candidate Aym stabilizing agent, to the signal generated in the presence of the candidate Aym stabilizing agent, and therefrom identifying Aym 15 stabilizing agent. In one embodiment the mitochondria are contained within cells, and in a further embodiment the agent that alters Aym is an agent that increases the level of cytosolic Ca2+. In another embodiment the agent that increases the level of cytosolic Ca2+ is a calcium ionophore or thapsigargin. In another embodiment the cells comprise one or more types of glutamate receptors. In another further embodiment the agent that 20 increases the level of cytosolic Ca2+ is an excitatory amino acid or a derivative thereof. In another further embodiment the excitatory amino acid or derivative thereof is glutamate, NAAG, NMDA, AMPA, APPA or kainate. In another embodiment the invention provides a Aym stabilizing agent identified according to the method just described. In another embodiment, the invention provides a method of treating stroke 25 comprising administering the Aym stabilizing agent to a patient in need thereof. These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

WO 00/79274 PCTUSOO/1 7380 19 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 schematically depicts direct and indirect methods for measuring energy transfer. Symbols: "XEX," peak excitation wavelength; "XEM," peak emission wavelength; "e," energy; open box, receptive filter setting; closed box, closed filter 5 setting. Figure 2 schematically depicts submitochondrial structural compartments and energy transfer interactions between energy transfer donor and acceptor molecules in designated compartments: "CS," cytosolic space; "OM," outer membrane; "IS," intermembrane space; "IM," inner membrane; "MX," matrix; "DMx," 10 donor compound localizing to the matrix; "AIM," acceptor compound localizing to the inner membrane; "DIS," donor compound localizing to the intermembrane space; "e," energy. Figure 3 shows representative data from FRET-based assays of Aym. Fig. 3A, data from a Type I assay; Fig. 3B, data from a Type II assay. 15 Figure 4 shows titration of an ET donor molecule (NAO) and an ET acceptor molecule (TMRM) in FRET assays of Aym. Figure 5 shows calibration of the concentrations of an ET donor molecule (NAO) and an ET acceptor molecule (TMRM) in FRET assays of Axpm. Figure 6 shows time-course data from a FRET assay of Ay, using NAO 20 and TMRM alone and in combination. Figure 7 shows Type I FRET AJm assay using various agents. Symbols: "MO," media (HBSS) only; "C," CCCP; "I," ionomycin; "I+BKA," ionomycin and bongrekic acid. Figure 8 shows Type I FRET Aym assay of various agents. Symbols: 25 "MO," media (HBSS) only; "C," CCCP; "I+RR," ionomycin and ruthenium red. Figure 9 shows Type I FRET Aym assay of various agents. Symbols: "MO," media (HBSS) only; "I," ionomycin; "I+CsA," ionomycin and cyclosporin A. The vertical lines indicate the standard error for each reading. Figure 10 is a dose-response curve for the Ay collapsing agent CCCP.

WO 00/79274 PCT/USOO/17380 20 Figure 11 shows Type II FRET Aym assay. Symbols: "MO," results from samples treated with media (HBSS) only; "4BA;" results from samples treated with the Aym-dissipating agent 4-bromo-A23187; "C," and arrow indicate time of CCCP addition to samples. 5 Figure 12 is a dose response curve for a Ay-dissipating compound (ionomycin). Figure 13 is a dose response curve for a compound (cyclosporin A) that protects mitochondria against a Ay-dissipating compound (ionomycin). Figure 14 shows a dose-response curve of three cell lines to the Aym 10 dissipating agent A-23187. Figure 15 shows FRET in carcinoma cells following experimentally induced loss of mitochondrial membrane potential. Figure 16 shows the concentration-dependent response of permeabilized cells to calcium ions, which leads to a collapse of Ay at higher concentrations of Ca*. 15 Figure 17 shows the same data presented in Figure 16 wherein mean values are plotted without error bars. Also, the response of permeabilized cells to CCCP, an agent known to induce Ay collapse, is not shown in Figure 16 but is presented here. It is noteworthy that, at a concentration of 100 uM, Ca induces collapse of Ay in a manner that is roughly equivalent, in terms of both the extent of 20 response and time course, to that seen in cells treated with CCCP. Figure 18 is a concentration response curve (CRC) of Ca2+ in permeabilized cells that was generated from the data presented in Figures 16 and 17. Figure 19 is a CRC of RU-360, an inhibitor of the mitochondrial calcium uniporter, in permeabilized cells that were also contacted with Ca2+ 25 Figure 20 is a CRC of cyclosporin A, an agent known to modulate Ca2+_ induced Ay collapse, in permeabilized cells that were also contacted with Ca2. Figure 21 shows a CRC of oligomycin, a specific ATP synthase inhibitor, in permeabilized SH-SY5Y cells. Figure 22 shows a CRC of ADP in permeabilized SH-SY5Y cells.

WO 00/79274 PCT/USOO/17380 21 Figure 23 shows a CRC of bongkrekic acid in permeabilized SH-SY5Y cells. Figure 24 shows a CRC of nigericin in permeabilized SH-SY5Y cells. SYMBOLS AND ABBREVIATIONS 5 Descriptions of specialized terms and abbreviations are listed in Table 1. Unless otherwise, indicated, symbols for nucleotides and amino acids are as described in 37 § C.F.R. 1.821. Term or I hmclo ntuet Abbreviation Description or Formula If Chemical or Instrument: (if any) Name of Supplier(s)* AT, ATm mitochondrial membrane potential -- ApH pH potential -- A-23187 1-(4,5-dimethoxy-2-nitrophenyl)ethyl Calbiochem ester 4-BA 4-bromo A-23 187 Calbiochem ANT adenine nucleotide translocator -- AO acridine orange MP ATR atractyloside Sigma BKA bongkrekic acid Biomol, Calbiochem --- BODIPY@ TR ceramide MP --- BODIPY@ FL Br 2

C

5 -ceramide MP --- BODIPY@ FL C 5 -ceramide MP --- BODIPY@ FL C 5 - sphingomyelin MP --- BODIPY® FL conjugate isomer 1 MP BFA brefeldin A from Penicillium MP brefeldianum calcein (a.k.a. fluorexon, fluorescein MP, Sigma complexon) WO 00/79274 PCTUSOO/17380 22 Term or If Chemical or Instrument: Abbreviation Description or Formula Name of Supplier(s)* (if any) CATR carboxyatractyloside Calbiochem CO-Fluro 5-carboxyfluorescein MP CCCP carbonyl cyanide m-chlorophenyl- Sigma hydrazone CsA cyclosporin A Calbiochem DAPI 4',6-diamidino-2-phenylindole MP DASPEI 2-(4-(dimethylamino)styryl)-N- MP ethylpyridinium iodide DASPMI dimethylaminostyrylmethylpyridinium MP iodide; comprises 2 isomers, 2-Di-1 ASP and 4-Di-1-ASP 2-Di-1-ASP 2-(4-(dimethylamino)styryl)-N- MP methylpyridinium iodide 4-Di-1-ASP 4-(4-(dimethylamino)styryl)-N- MP methylpyridinium iodide Di1C 1 6(3) 1,1 '-dihexadecyl-3,3 ,3' ,3'-tetramethyl- MP indocarbocyanine perchlorate Di1C 1 8(3) 1,1 '-dioctadecyl-3,3,3 ',3'-tetramethyl- MP indocarbocyanine perchlorate --- 4-dimethylamino-tetramethylrosamine MP DiOC 2 (5) 3,3'-diethyloxadicarbocyanine iodide MP DiOC 5 (3) 3,3'-dipentyloxacarbocyanine iodide MP DiOC 6 (3) 3,3'-dihexyloxadicarbocyanine iodide MP DiOC 7 (3) 3,3'-diheptyloxadicarbocyanine iodide MP EtBr ethidium bromide Sigma ET energy transfer

---

WO 00/79274 PCTUSOO/17380 23 Term orIf Chemical or Instrument: Abbreviation Description or Formula IfC e or Instrument: Name of Supplier(s)* (if any) ETC electron transport chain -- FCCP carbonyl cyanide p- S i g ma (trifluoromethoxy)phenyl-hydrazone FLASH fluorescein arsenical helix binder --

FLIPR

TM Fluorometric Imaging Plate Reader Mol. Dev. FRET fluorescence resonance energy transfer --

FUN-I

TM (proprietary compound) MP --- hydroxystilbamidine, methanesulfonate MP JC-1 5,5',6,6'-tetrachloro-1,1',3,3'-tetra- MP ethylbenzimidazoylcarbocyanine iodide lucigenin bis-N-methylacridinium nitrate MP LysoSensor TM (proprietary compounds) MP s LysoTracker (proprietary compounds) MP TMS MELAS Mitochondrial Encephalopthy, Lactic -- Acidosis and Stroke MixCon Mixed Controls (cybrids) -- MPP+ I -methyl-4-phenylpyridinium Calbiochem, RBI MPT Mitochondrial Permeability Transition mtDNA mitochondrial DNA -- MitoFluorTMs (proprietary compounds) MP MitoTracker (proprietary compounds) MP @s NAO 1 O-N-nonyl acridine orange MP --- NBD C 6 -ceramide MP WO 00/79274 PCTUSOO/17380 24 Term orIf Chemical or Instrument: iAbbreviation Description or Formula IfC e or Instrument: (if any)Name of Supplier(s)*~ (if any) --- NBD C 6 -sphingomyelin MP -- - oligomycin Calbiochem PI propidium iodide S i g ma PMF protonmotive force rh123 rhodamine 123 MP, Calbiochem rhB rhodamine B MP rh6G rhodamine 6G MP RR ruthenium red (ammoniated ruthenium Sigma oxychloride) SNAFL@ seminapthofluorescein calcein MP calcein SYTO@ 18 (proprietary compound) MP TB-rhl23 tetrabromorhodamine 123 MP TMRE tetramethylrhodamine, ethyl ester MP TMRM tetramethyirhodamine, methyl ester MP --- tetramethylrosamine MP --- 4-dimethylaminotetramethylrosamine MP --- thapsigargin Calbiochem --- valinomycin Calbiochem Green Fluorescent Proteins vectors from Aurora / Clontech GFP green fluorescent protein -- BFP blue-shifted green fluorescent protein -- CFP cyan-shifted green fluorescent protein -- RFP red-shifted green fluorescent protein -- YFP yellow-shifted green fluorescent protein --- WO 00/79274 PCTUSOO/17380 25 * Abbreviations for suppliers: "Calbiochem", Calbiochem, Inc., La Jolla, CA; "MP," Molecular Probes, Inc., Eugene, OR; "Biomol," Biomol Research :Laboratories, Inc., Plymouth Meeting, MA; "Mol. Dev.," Molecular Devices, Sunnyvale, CA; "Aurora," Aurora Biosciences Corp., San Diego, CA; "Clontech," CLONTECH Laboratories, Inc., 5 Palo Alto, CA; "Sigma," Sigma Chemical Co., St. Louis, MO; RBI, Research Biochemicals International, Natick, MA. DETAILED DESCRIPTION OF THE INVENTION The present invention pertains in part to the use of intermolecular energy transfer to monitor intracellular and intraorganellar conditions. In particular, the 10 invention derives from the unexpected observation that such intracellular and intraorganellar conditions can be surveyed using energy transfer molecule donor acceptor pairs that need not undergo specific intermolecular recognition events such as affinity binding interactions. Rather, according to the present disclosure, under particular naturally occurring or artificially induced intracellular and/or intraorganellar 15 physiologic conditions, appropriately paired energy transfer donor and acceptor molecules can be selected that accumulate at acceptably adjacent sites as provided herein, to generate detectable signals. By way of background, energy transfer (ET) is generated from a resonance interaction between two molecules: an energy-contributing "donor" molecule 20 and an energy-receiving "acceptor" molecule. Energy transfer can occur when (1) the emission spectrum of the donor overlaps the absorption spectrum of the acceptor and (2) the donor and the acceptor are within a certain distance (for example, less than about 10 nm) of one another. The efficiency of energy transfer is dictated largely by the proximity of the donor and acceptor, and decreases as a power of 6 with distance. 25 Measurements of ET thus strongly reflect the proximity of the acceptor and donor compounds, and changes in ET sensitively reflect changes in the proximity of the compounds such as, for example., association or dissociation of the donor and acceptor. According to the present invention, both energy transfer molecules, the ET donor molecule and the ET acceptor molecule, are molecules that are not WO 00/79274 PCT/USOO/17380 26 endogenous to the sample as provided herein (by way of non-limiting example, a cell, an organelle such as a mitochondrion, or a subcellular or suborganellar compartment) with which they are contacted. The donor and acceptor compounds may co-localize to a subcellular compartment in such a manner as to achieve sufficient proximity to one 5 another for a particular type of energy transfer to occur. In certain aspects of the invention, such co-localization may be dependent upon, or may be disrupted by, intracellular processes or responses to chemical agents. For instance, such processes or responses can lead to, respectively, an increase or a decrease in energy transfer that can be detected, for example, by detecting a signal. Thus, for example, detection of the 10 degree or rate of energy transfer between the ET donor and ET acceptor molecules may provide in pertinent part a method for assaying a given intracellular process or response. In certain preferred embodiments the invention provides a method for assaying a cellular membrane potential, and in certain other preferred embodiments the invention provides a method for assaying mitochondrial membrane potential. 15 It is therefore an aspect of the invention to provide a method for assaying a cellular membrane potential, in pertinent part, by contacting a sample comprising one or more cellular membranes with an ET donor and an ET acceptor molecule, exciting the ET donor to produce an excited ET donor molecule and detecting a signal generated by energy transfer from the ET donor to the ET acceptor. The sample may be contacted 20 with the ET donor and the ET acceptor simultaneously, or it may be contacted with the ET donor and the ET acceptor sequentially and in either order, depending on the particular donor and acceptor being used. Optionally, the sample may be washed under suitable conditions prior to the step of detecting a signal, for example to improve sensitivity for detecting the signal. Those having ordinary skill in the art can readily 25 determine the manner by which the sample is contacted, in view of the properties of the sample and of the ET molecules selected, and in view of the teachings provided herein. As also provided herein, the subject invention method can employ any suitable ET donor molecule and ET acceptor molecule that can function as a donor-acceptor pair. As discussed in greater detail below, the method of the present invention may be used to WO 00/79274 PCT/USO0/17380 27 identify an agent that alters a cellular membrane potential, or to identify a molecule that is a regulator of such an agent. In certain preferred embodiments the invention is directed to a method for assaying mitochondrial membrane potential, wherein neither the ET donor molecule 5 nor the ET acceptor molecule is endogenous to mitochondria, and wherein the ET donor and the ET acceptor each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites as provided herein. Optionally, in preferred embodiments the ET donor molecule and the ET 10 acceptor molecule may both be light emission molecules, for example fluorescent, phosphorescent, or chemiluminescent molecules or the like, which emit a detectable signal in the form of light when excited by excitation light of an appropriate wavelength. Preferred ET donor-acceptor combinations that can be used according to the present invention are fluorescent donors with fluorescent or phosphorescent 15 acceptors, or phosphorescent donors with phosphorescent or fluorescent acceptors. "Fluorescence" refers to luminescence (emission of light) that is caused by the absorption of radiation at one wavelength ("excitation"), followed by nearly immediate re-radiation ("emission"), usually at a different wavelength, that ceases almost at once when the incident radiation stops. At a molecular level, fluorescence occurs as certain 20 compounds, known as fluorophores, are taken from a ground state to a higher state of excitation by light energy; as the molecules return to their ground state, they emit light, typically at a different wavelength. "Phosphorescence," in contrast, refers to luminescence that is caused by the absorption of radiation at one wavelength followed by a delayed re-radiation that occurs at a different wavelength and continues for a 25 noticeable time after the incident radiation stops. "Chemiluminescence" refers to luminescence resulting from a chemical reaction, and "bioluminescence" refers to the emission of light from -living organisms or cells, organelles or extracts derived therefrom. In certain preferred embodiments, a detectable signal that is generated by 30 energy transfer between ET donor and acceptor molecules results from fluorescence WO 00/79274 PCT/USOO/17380 28 resonance energy transfer (FRET). FRET occurs within a molecule, or between two different types of molecules, when energy from an excited donor fluorophore is transferred directly to an acceptor fluorophore (for a review, see Wu et al., Analytical Biochem. 218:1-13, 1994). In general, the energy transfer from an excited fluorophore 5 (e.g., an ET donor molecule) to an absorber (e.g., an ET acceptor molecule) is measured by (1) measuring the spectra (including changes in the spectra) of fluorescence from the energy donor molecule and the energy acceptor molecule; (2) measuring the speed at which the intensity of the fluorescent intensity of the energy donor molecule decreases after pulse-laser excitation (i.e., the fluorescence lifetime); or (3) measuring the 10 reduction in intensity of fluorescence from the energy donor compound (i.e., indirect measurement of FRET), or the increase in intensity of fluorescence from the energy acceptor compound (i.e., direct measurement of FRET). Direct measurement of energy transfer involves monitoring the signal from an excited energy acceptor molecule, which increases as the ET compounds achieve proximity to each other, whereas indirect 15 measuring of energy transfer involves monitoring a signal from an excited ET donor molecule that decreases (i.e., that is quenched) as the compounds achieve proximity (Figure 1). The use of FRET to monitor specific intermolecular and/or intramolecular interactions that involve specific inter- and intramolecular recognition 20 events (including associative and dissociative events, e.g., affinity and binding interactions) that bring ET donor and ET acceptor fluorophores into close proximity with one another, is known in the art. When measuring such intermolecular interactions, the ET donor and acceptor fluorophores are typically situated on two different molecules that are known or believed to enter into close association with each 25 other. On the other hand, when intramolecular interactions are measured, the ET donor and acceptor fluorophores are present on the same molecule. In contrast to such known uses of FRET methodologies, wherein ET donor and acceptor fluorophores are brought into proximity with each other through known specific molecular interactions, the present invention is based on the unexpected 30 observation that energy transfer can occur between ET donor and ET acceptor WO 00/79274 PCT/USOO/17380 29 fluorophores that are brought into proximity with one another by virtue of their having selectively concentrated or accumulated in a common subcellular compartment, for example, an organelle, a sub organellar site or other subcellular locale. As a result, the present invention can be used to monitor a variety of conditions or processes within, or 5 associated with, such subcellular compartments. As provided herein, contemplated uses of the invention include but need not be limited to (i) monitoring conditions and processes within subcellular compartments, (ii) monitoring interactions between pairs of macromolecules found within or associated with such subcellular compartments, (iii) identifying agents that 10 influence subcellular compartments and/or intracellular processes in a species-specific manner, and (iv) identifying agents that influence subcellular compartments and/or intracellular processes in such a manner as to treat diseases and disorders of mammals and other animals, including humans, and plants. Each of these uses is described in greater detail below. 15 Typically, the invention relates in part to a method for assaying a sample, which in preferred embodiments is a biological sample and in particularly preferred embodiments is a biological sample containing one or more mitochondria. In other preferred embodiments the biological sample contains one or more cellular membranes, including the plasma membrane and intracellular membrane bounded 20 compartments such as endosomes, lysosomes, peroxisomes, mitochondria, chloroplasts, endocytic and secretory vesicles, ER-Golgi constituents, organelles and the like. Biological samples may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source. The subject or biological source may be a human or non-human 25 animal, a plant, a unicellular or a multicellular organism, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid "cybrid" cell lines, differentiated or differentiatable cell lines, 30 transformed cell lines and the like.

WO 00/79274 PCT/USOO/17380 30 In certain embodiments of the invention, it may be preferred to use intact cells whereas, in certain other embodiments, the use of permeabilized cells may be preferred. A permeabilized cell is a cell that has been treated in a manner that results in a partial or complete loss of plasma membrane selective permeability. As a first 5 example, it may be desirable to permeabilize a cell in a manner that permits calcium cations in the extracellular milieu to diffuse into permeabilized cells and contact mitochondria. Thus, in this instance, permeabilization serves as an alternative to the use of a calcium ionophore. As a second example, certain detectably labeled molecules, such as certain of the ET donor and/or ET acceptor molecules provided herein, may 10 penetrate the plasma membrane at a moderate rate, or to a limited degree, unless their entry into the cytosol is facilitated in some manner. Permeabilization of cells is one manner by which the cytosolic entry of such ET molecules can be facilitated. As a third example, some candidate agents being tested according to the method may penetrate the plasma membrane at a moderate rate, or to a limited degree, unless their entry into the 15 cytosol is facilitated in some manner. Permeabilization of cells is one manner by which the entry of such candidate agents into the cytosolic space can be facilitated. Active agents that are identified under these conditions can subsequently be modified chemically to enhance their uptake by whole cells; active agents that are so modified are expected to serve as lead compounds for drug development and, in some instances, may 20 themselves be used as drugs or as drug candidates. Those having ordinary skill in the art are familiar with methods for permeabilizing cells, for example by way of illustration and not limitation, through the use of surfactants, detergents, phospholipids, phospholipid binding proteins, enzymes, viral membrane fusion proteins and the like; by exposure to certain bacterial toxins, 25 such as a-hemolysin; by contact with hemolysins such as saponin (which is also a nonionic detergent, as is digitonin); through the use of osmotically active agents; by using chemical crosslinking agents; by physicochemical methods including electroporation and the like, or by other permeabilizing methodologies including, e.g., physical manipulations such as electroporation. Those skilled in the art are familiar 30 with methods for permeabilizing cells and can readily determine without undue WO 00/79274 PCT/USOO/17380 31 experimentation the most appropriate permeabilizing agent for use according to the present invention as provided herein. Relevant factors for this determination include but are not limited to toxicity of the permeabilizing agent to a specific cell, the molecular size of the molecule for which entry into the cell is sought through the use of 5 permeabilization, and the like (see, e.g., Schulz, Methods Enzymol. 192:280-300, 1990). Thus, for instance, cells may be permeabilized using any of a variety of known techniques, including addition of permeabilizing agents such as bacterial toxins, for example, streptolysin 0, Staphylococcus aureus a-toxin (a.k.a. a-hemolysin); other hemolytic agents such as saponin; or exposure to one or more detergents (e.g., 10 digitonin, Triton X-100, NP-40, n-Octyl p-D-glucoside and the like) at concentrations below those used to lyse cells and solubilize membranes (i.e., below the critical micelle concentration). Certain common transfection reagents, such as DOTAP, may also be used. ATP can also be used to permeabilize intact cells, as may be low concentrations of chemicals commonly used as fixatives (e.g., formaldehyde). All of the 15 permeabilizing agents described in this paragraph are available from, e.g., Sigma Chemical Co., St. Louis, MO (see Sigma catalog entitled "Biochemicals and Reagents for Life Science Research," Anon., 1999, and references cited therein for these and other permeabilizing agents). In certain embodiments of the invention, the subject or biological source 20 may be suspected of having or being at risk for having a disease associated with organellar dysfunction including altered mitochondrial function and mitochondrial dysfunction, and in certain embodiments of the invention, the subject or biological source may be known to be free of a risk or presence of such a disease. Organellar dysfunction may further include abnormal, supranormal, inefficient, ineffective or 25 deleterious activity at the organelle level, for example, defects in uptake, release, activity, sequestration, transport, metabolism, catabolism, synthesis, storage or processing of biological molecules and macromolecules such as proteins and peptides and their derivatives, carbohydrates and oligosaccharides and their derivatives including glycoconjugates such as glycoproteins and glycolipids, lipids, nucleic acids and 30 cofactors including ions, mediators, precursors, catabolites and the like. Examples of WO 00/79274 PCTIUSOO/17380 32 organellar dysfunction may include, but need not be limited to, lysosomal storage defects such as the mucopolysaccaridoses, I-cell disease, Wolman disease and cholesteryl ester storage disease (e.g., Du et al., 1998 Mol. Genet. Metab. 64:126-34); plasma membrane defects such as ion channel dysfunction in cystic fibrosis; 5 endoplasmic reticulum storage diseases (e.g., Kim and Arvan, 1998 Endocr. Rev. 19:173-202); diseases associated with Golgi defects (e.g., ALS, AD, Gonatas et al., 1998 Histochem. Cell. Biol. 109:591-600) and other types of organellar dysfunction that are known to those familiar with the art. In certain preferred embodiments it may be desirable to compare the 10 signal detected according to the method of the invention with a reference signal. Selection of a suitable reference signal will according to criteria with which those having ordinary skill in the art will be familiar, and may vary depending on the particular cellular membrane being assayed and upon the particular donor-acceptor pair employed. For example, a reference signal may be generated by a reference compound 15 such as an ET donor or ET acceptor molecule or a distinct reporter molecule that is an indicator as provided herein, and may further be generated in the absence or presence of a sample. Such reporter molecules or indicators may include a detectable compound that can be detected as indicative of one or more of a quantity of a detectable component or a location of a detectable component, or the like. For example, by way of 20 illustration and not limitation, a reference signal may be generated by a reporter molecule that permits normalization of a detected energy transfer signal according to the number of cells present (e.g., the reporter may be any of numerous known indicators of cell number, such as selective stains for cell nuclei, for example, propidium iodide or ethidium bromide). 25 In certain other embodiments, the reference signal is generated by an indicator of the mitochondrial mass, the mitochondrial number or the mitochondrial volume present. For example, where an indicator of mitochondrial mass is selected, a reporter molecule such as nonylacridine orange (which can also be an ET donor) may be employed. Methods for quantifying mitochondrial mass, volume and/or 30 mitochondrial number are known in the art, and may include, for example, quantitative WO 00/79274 PCTIUSOO/17380 33 staining of a representative biological sample. Typically, quantitative staining of mitochondrial may be performed using organelle-selective probes or dyes, including but not limited to mitochondrion selective reagents such as fluorescent dyes that bind to mitochondrial molecular components (e.g., nonylacridine orange, MitoTrackersTM) or 5 potentiometric dyes that accumulate in mitochondria as a function of mitochondrial inner membrane electrochemical potential (see, e.g., Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes, Eugene, OR). As another example, mitochondrial mass, volume and/or number may be quantified by morphometric analysis (e.g., Cruz-Orive et al., 1990 Am. J Physiol. 10 258:L148; Schwerzmann et al., 1986 J Cell Biol. 102:97). These or any other means known in the art for quantifying mitochondrial mass, volume and/or mitochondrial number in a sample are within the contemplated scope of the invention. For example, the use of such quantitative determinations for purposes of calculating mitochondrial density is contemplated and is not intended to be limiting. In certain highly preferred 15 embodiments, mitochondrial protein mass in a sample is determined using well known procedures. For example, a person having ordinary skill in the art can readily prepare an isolated mitochondrial fraction from a biological sample using established cell fractionation techniques, and therefrom determine protein content using any of a number of protein quantification methodologies well known in the art. 20 In other embodiments, a reference signal may be generated by a reporter molecule that permits normalization of a detected energy transfer signal according to the amount of protein present (e.g., coomassie blue, fluorescamine, bicinchoninic acid) or to the amount of nucleic acid present (e.g., ethidium bromide, acridine orange, methylene blue). As another example, a reference signal may be generated by a 25 detectable reporter molecule that is soluble in a liquid medium containing the sample, but that cannot traverse cellular membranes and so serves as a marker of extracellular medium, for example as an indicator of fluid volume. For example, where extraordinarily sensitive instrumentation (e.g., see infra) may be used to detect ET signals, such an indicator may permit improved quantitative precision by calibration/ 30 normalization of sample volumes. Many compounds that are suitable for use as such WO 00/79274 PCT/USO0/17380 34 reference signals will be known to those familiar with the art, who may select such compounds as sources of a reference signal in a manner dependent on, inter alia, the particular cellular membrane potential being assayed and the particular donor-acceptor pair employed. 5 As used herein, detecting a "relative amount" of a signal may include but is not limited to detecting a signal for purposes of comparing it to a reference signal as provided above. Thus, detecting a relative amount of a signal may refer to detecting only a portion of a signal (e.g., detecting a signal at less than 100% efficiency), or to detecting a signal only a portion of which is generated by energy transfer, or to 10 detecting a portion of a signal relative to a signal detected from another sample such as a control sample, regardless of whether any of such other signals detected are reference signals as provided herein. Detection of a signal according to the methods disclosed herein may include quantification of ET by conventional or arbitrarily assigned units of measure. In certain embodiments, a signal may be detected over a period of time such 15 that one or more behaviors of the signal may be analyzed as a function of time. For instance, in some embodiments described herein, a signal may be detected over a period of time, which refers to any method of detecting a sample in a manner that provides more than a single detection event, such that a correlation of a detected signal with a discrete point in time can be established. Thus, for example, in certain embodiments a 20 change in an amount of a signal may be detected over two or more time points, and a rate of change in the level of signal is determined (e.g., a slope or a rate-of-change of a slope such as a first order derivative is determined, when the signal level is plotted as a function of time). As another example, in certain other embodiments an amount of a signal may be cumulatively determined over a discrete time interval, to provide a 25 summed signal (e.g., an integrated signal). These and other techniques known in the art for analyzing quantitative data, and in particular for analyzing such data having a temporal component, are within the contemplated invention and are described in greater detail below. Thus, any of the methods provided by the invention can be modified so 30 as to also include a reference signal that correlates with a reference parameter of interest WO 00/79274 PCT/USOO/17380 35 for the purpose of, e.g., standardizing for cell number, quantity of cellular protein or cellular nucleic acids, mitochondrial mass, quantity of mitochondrial protein or mitochondrial nucleic acids, indicator of fluid volume or the like. The reference signal, which can be used as an internal standard, need not result from energy transfer and can 5 involve any signal that can be correlated with the desired reference parameter but which does not interfere with detection of the test/assay signal. In the context of the invention, a reference compound can interfere with the test/assay signal if it generates a signal that cannot be resolved from the test/assay signal, or if it localizes to the same subcellular compartment as the ET donor and acceptor compounds and itself acts as an ET acceptor 10 or donor compound. An instrument such as FLIPRTM can be set to alternate between reading signals at two different wavelengths with a cycling time of about one second; in this manner, the reference signal and the test/assay signal (e.g., FRET, Ay) can be read over the same time course. However, the reference need not be read at the same time as the 15 test/assay signal. For example, in some aspects of the invention, it is necessary to disrupt the cells in order to detect the reference signal, and this typically necessitates that the reference signal be read after the test or assay has been completed. Some non-limiting examples of reference signals include the following. After the test or assay, as is known in the art, cellular protein (including mitochondrial 20 protein) can be measured using methods such as the Bradford or Lowry assays, and nucleic acid can be measured via the use of fluorescent dyes such as propidium iodide (PI). Nucleic acids can also be measured in living cells. For example, in digitonin permeabilized cells, propidium iodide (PI; peak excitation, 536 nm; peak emission, 617 nm when bound to a nucleic acid) binds nuclear and cytoplasmic nucleic acids but 25 cannot access the mitochondrial matrix and the mitochondrial nucleic acids contained therein; PI thus provides a reference signal for quantity of cellular nucleic acids. The permeant compound acridine orange (AO) can be used in living cells to distinguish RNA and DNA as it has distinct excitation/emission spectra depending on the type of nucleic acid to which it is bound (AO:DNA, peak excitation, 500 nm; peak emission, 30 526 nm; AO:RNA, peak excitation, 460 nm; peak emission, 650 nm). The SYTO stains WO 00/79274 PCT/USOO/17380 36 can also be used to detect nucleic acids in living cells; the manufacturer (Molecular Probes, Inc., Eugene, OR) of the SYTO stains indicates that all of the SYTO stains can access nuclear and cellular nucleic acids and some can also access mitochondrial nucleic acids; one skilled in the art will be able to apply techniques such as, e.g., 5 fluorescent microscopy to determine what types of nucleic acids are detected by the use of a particular SYTO stain. JC- 1 green fluorescence and NAO fluorescence can be used to measure mitochondrial mass in living cells (Mancini et al., Ann. Surg. Oncol. 5:287 295, 1998; Vayssiere et al., In Vitro Cell. Dev. Biol. 28A:763-772, 1992, respectively). The present invention provides diagnostic and prognostic methods, as 10 well as screening assays, i.e., methods of identifying agents that alter (i.e., increase or decrease in a statistically significant manner) a monitored process or condition, for example mitochondrial membrane potential. Diagnostic uses include methods for assaying a cellular process or condition (e.g., a cellular membrane potential such as mitochondrial membrane potential) wherein a biological sample comprising a cellular 15 membrane or subcellular compartment (e.g., an organelle such as a mitochondrion) is taken from a patient suspected of having or being prone or predisposed to a disease or disorder (e.g., having an increased risk for or probability of developing the disease relative to the risk in a reference population), and wherein further the process or condition may be altered relative to that determined in a control sample derived from a 20 patient known to not have the disease or disorder. Prognostic uses include methods wherein a biological sample comprising a cellular membrane or subcellular compartment is taken from a patient known to have a disease or disorder in which the monitored intracellular process or condition is altered. In such prognostic uses, for example, biological samples from the patient are prepared and tested for their response 25 to agents known to impact the monitored intracellular process or condition in some, but not all, instances. A desired response of the biological sample to a particular agent indicates that the patient from which the sample was taken will respond best to a treatment that correlates with positive response to that treatment. In a related aspect, pharmacogenetic studies using the invention are employed to determine the correlations 30 between different treatments and specific measurements generated by the invention.

WO 00/79274 PCT/USOO/17380 37 Non-limiting examples of diseases or disorders that are thought to involve the altered function or dysfunction of subcellular compartments include Alzheimer's disease, Parkinson's disease, type II diabetes and lysosomal storage disorders. When the subcellular compartment of interest is the mitochondrion, 5 preferred biological samples are cybrids (e.g., cytoplasmic hybrid cells comprising a common nuclear component but having mitochondria derived from different individuals, i.e., patients and controls). Methods for preparing and using cybrids are described in U.S. Patent No. 5,888,438, published PCT applications WO 95/26973 and WO 98/17826, King and Attardi (Science 246:500-503, 1989), Chomyn et al. (Mol. 10 Cell. Biol. 11:2236-2244, 1991), Miller et al. (J. Neurochem. 67:1897-1907, 1996), Swerdlow et al. (Annals of Neurology 40:663-671, 1996), Cassarino et al. (Biochim. Biophys. Acta 1362:77-86, 1997), Swerdlow et al. (Neurology 49:918-925, 1997), Sheehan et al. (J. Neurochem. 68:1221-1233, 1997), and Sheehan et al. (J. Neurosci. 17:4612-4622, 1997), all of these being hereby incorporated by reference. 15 The term "screening" refers to the use of the invention to identify agents that impact the monitored intracellular process or condition in a negative or positive fashion. Cells or organelles are treated with an agent thought to impact the monitored intracellular process or condition, and the response of a subcellular compartment of interest to the agent is monitored and compared to a control sample that has been treated 20 with only the vehicle used to deliver the agent. Agents that impact the monitored intracellular process or condition result in an altered response of the subcellular compartment of interest relative to the response in the control sample. In certain aspects of the invention, agents that act in a species-specific manner are identified by the screening methods of the invention. 25 The present invention relates to energy transfer between chemically distinct and independent ET donor and acceptor molecules that can occur (i) when both ET donor and ET acceptor molecules are localized to the same subcellular compartment; (ii) when one ET molecule (i.e., the ET donor or the ET acceptor) is localized to a particular subcellular compartment and the other ET molecule (i.e., the 30 ET acceptor or the ET donor) is localized to a membrane that forms one border of that WO 00/79274 PCT/USOO/17380 38 subcellular compartment; or (iii) when one ET molecule (i.e., the ET donor or the ET acceptor) is localized to a subcellular compartment and the other ET molecule (i.e., the ET acceptor or the ET donor) transiently or otherwise associates with that subcellular compartment. 5 In situation (i), a change in the efficiency and/or rate of energy transfer between the ET donor and acceptor molecules correlates with a change in a condition or the occurrence of a given process within the subcellular compartment of interest. Non limiting examples of this aspect of the invention, described in greater detail below, include methods for assaying mitochondrial membrane potential (AT) or pH potential 10 (ApH), photosynthesis within chloroplasts, and formation of secondary lysosomes. According to the invention such methods may also be used to detect the presence of specific cell types in a biological sample, when at least one subcellular compartment of a specific cell type accumulates and/or retains the ET donor or acceptor molecule to a greater extent than do other cell types. 15 In situation (ii), a change in the rate of energy transfer between the ET donor and acceptor molecules correlates with a process that influences the cellular membrane (e.g., alters the membrane potential) containing either the ET donor or ET acceptor molecule, and/or influences the subcellular compartment bounded by the cellular membrane, which compartment contains the other member (e.g., ET acceptor or 20 donor molecule) of the ET molecule pair. Non-limiting examples of this aspect of the invention, described in more detail herein, include methods for monitoring the mitochondrial pore transition (MPT) and viral uncoating processes. In situation (iii), a change in the rate of energy transfer between the ET donor and acceptor molecules correlates with the association of a detectably labeled 25 molecule (e.g., labeled with either an ET donor or ET acceptor) with, or its dissociation from, a labeled subcellular compartment (e.g., labeled with either an ET acceptor or ET donor). Non-limiting examples of such embodiments of the invention, described in greater detail below, include methods for monitoring the association of Bcl-2 protein with, or the dissociation of cytochrome c from, the outer mitochondrial membrane.

WO 00/79274 PCT/USOO/17380 39 Donor-Acceptor Pairs There are provided, according to the present invention, paired ET molecules wherein each pair comprises an ET donor molecule and an ET acceptor molecule. As described herein there are several criteria for determining combinations 5 of energy-donating compounds (ET donor molecules) and energy-accepting compounds (ET acceptor molecules) that are acceptable for ET-based assays of the invention. Additional criteria may specifically apply when the assay is designed to monitor a particular intracellular state or activity such as, for example, mitochondrial inner membrane potential (Ay or Aym), association of a particular intracellular molecule or 10 factor with a particular organelle, release of a particular intracellular molecule or factor from an organelle or the like. One criterion for determining a suitable ET donor-acceptor pair for use according to the present invention is that the energy emission spectrum of the ET donor molecule should at least partially overlap the energy absorption spectrum of the ET 15 acceptor molecule, so that energy transfer from the donor to the acceptor can occur. Typically, an ET donor compound has an emission peak wavelength (herein, "XD(em)") that is within several nm of the excitation peak wavelength of the acceptor compound (herein, "kA(ex)"). That is, the difference between D(em) and A(ex) is typically from about 70 nm to about 20 nm or less, with typical values for the difference 20 A = kD(em) - kA(ex) being <60 nm, <50 nm, <40 nm, 30 nm, <25 nm, 20 nm, <15 nm, <10 nm, <5 nm or <1 nm. When excitation or emission is plotted as a function of wavelength, however, 25 certain compounds that are suitable for use as ET donor molecules or ET acceptor molecules may have broad peaks, such that energy may be detectably transferred between certain paired ET donor and ET acceptor molecules having a larger difference between D(em) and A(ex) than that just described. For example, certain donor-acceptor pairs may be suitable for ET methodologies as provided herein even where energy 30 transfer between them is highly inefficient (i.e., where one or both of the ET donor and acceptor may be used with light having a wavelength that is far from the excitation peak WO 00/79274 PCTUSOO/17380 40 wavelength and/or the emission peak wavelength for the ET molecule), so long as the ET donor and the ET acceptor are within sufficient proximity of one another for detectable energy transfer to occur. Those having ordinary skill in the art can readily determine without undue experimentation when fluorescence resonance energy transfer 5 is present, such that selection of appropriate ET donor-acceptor pairs may be accomplished according to established criteria and the teachings provided herein. For example, routine screening may be employed by combining in solution (e.g., in the absence of a biological sample) at least a candidate ET donor molecule and a candidate ET acceptor molecule as disclosed herein, for purposes of 10 determining whether a detectable FRET signal can be generated. For certain donor acceptor combinations, selective accumulation of one or both of the donor and acceptor in a subcellular compartment may depend on binding of the donor and/or the acceptor to a molecule present in the subcellular compartment, and for other donor-acceptor pairs accumulation in such compartments may not involve such binding. Thus, screening of 15 certain donor-acceptor pairs for their facilitation of a detectable FRET signal in solution may include adding to the solution at least one suitable biomolecule such as a protein or peptide-, a lipid-, a nucleic acid- or a carbohydrate-containing species that will be selected by the person having ordinary skill in the art based upon familiarity with the nature of the donor and/or the acceptor and/or the properties of a subcellular 20 compartment in a contemplated biological sample to be used in the subject invention method. Without wishing to be bound by theory, in order to detect a FRET signal the concentrations of the ET donor and acceptor molecules used in such a pilot experiment may in certain such instances exceed those to be used in the subject invention methods as provided herein. However, similarly detectable concentrations of such ET molecules 25 may accumulate in a sample subcellular compartment as described herein, even where substantially lower concentrations of ET molecules are initially contacted with the sample. Those familiar with the art will also readily appreciate that the fluorescence spectral properties of ET donor and ET acceptor molecules may vary as a function of solution and sample conditions employed (e.g., solvent selected, solvent and ionic 30 strength, pH, nature of the sample, etc.).

WO 00/79274 PCT/USOO/17380 41 Another criterion useful in selecting a suitable ET donor-acceptor pair for use according to the present invention is that the emission signal from the excited ET acceptor compound must be capable of being distinguished from the emission signal from the excited ET donor compound. An emission signal from an excited donor can 5 be so distinguished if, for example, (1) the wavelength of the emission signal from the excited acceptor is sufficiently distinct from the wavelength of the emission signal from the excited donor or (2) the acceptor quenches the emission signal from the excited donor. A variety of classes of compounds can serve as ET acceptor molecules 10 and ET donor molecules according to the present invention, and the acceptor and donor can, but need not, belong to the same class of compound. For instance, a fluorescent protein might serve as an ET donor molecule for an ET acceptor that is a small organic compound, or to an acceptor that is a different fluorescent protein, so long as other criteria necessary for the assay are satisfied. Table 1 lists, among other things, 15 abbreviations for ET donor and acceptor compounds, and Table 2 lists some ET donor acceptor pairings that are appropriate for ET-based assays (with the exception of the various Green Fluorescent Protein derivatives, most of the compounds listed in Table 2 are available from Molecular Probes, Inc., Eugene, OR).

WO 00/79274 PCT/USOO/17380 42 Table 2: Donor-Acceptor Pairs for ET-Based Assays DONORS ACCEPTORS Peak Peak Peak Peak Excitation Emission Excitation Emission Wave- Wave- Wave- Wave Compound length length length length Compound Group I 373 -388 400-500 Suitable for Use with Any Group I nm nm Donor: BFP- 380 nm 440 nm 433 nm 475 nm CFP F64L/S65T/ (501 nm)* F64L/S65T/ Y66H/Y145F Y66W/N1461/ M153T/ BFP-Y66H/ 381 nm 445 nm V163A/N212L Y145F BFP-Y66H 382 nm 448 nrn 461 nm 585 nm 2-Di-1-ASP BFP-F64M/ 385 nm 450 urn 461 nm 589 nm DASPEI Y66H/V681 LysoTracker TM 373 nm 422 urn 470 nrn* 510 urn wildtype Yellow DND- GFP 22 LysoSensor T M 374 nrn 424 nm 466 nm 536 nm NBD C 6 Yellow DND- ceramide 192 LysoSensorTM 373 nm 425 nm 466 nm 536 un NBD C 6 Yellow DND- sphingomyelin 167 475 urn 605 nm 4-Di-1-ASP 442 rnm 505 nrn LysoSensor TM Green DND 153 443 nm 505 nm LysoSensor TM Green DND 189 479 un 507 nm RFP-S65C WO 00/79274 PCTUSOO/17380 43 482 nm 504 nm DiOC 7 (3) 483 nm (none) SYTO 18 484 nm 501 nm DiOC 6 (3) 484 nm 500 nm DiOC 5 (3) 488 nm 507 nm RFP F64L/S65T 489 nm 511 m RFP-S65T 490 nm 509 nm RFP-F64M/ S65G/Q69L 485 -585 590 nm JC-1 nm aggregates** Group IIA 360 - 375 465 - 560 Suitable for Use with Any Group nm nm or IIC Donor: IIA, IIB DAPI 365 nm 520 nm 466 nm 536 nm NBD C 6 ceramide hydroxystilba- 361 nm 536 nm 466 nm 536 nm NBD C 6 midine, sphingomyelin methane sulfonate Group IIB 390-405 465 -560 475 nm 605 nm 4-Di-1-ASP nm nm wildtype 395 nm 510 nm 483 nm (none) SYTO@ 18 GFP (470 n)* 484 nm 500 nm DiOC 5 (3) 502 nm 512 nm YFP S65G/Y66W/ S72A/T203Y 503 nm 510 nm Brefeldin A, BODIPY@ FL conjugate isomer I WO 00/79274 PCTUSOO/17380 44 Group IIC 445 - 460 465 - 560 507 nm 529 nm rhodamine 123 nm nm Lucigenin 455 rn 505 n 510 nm 527 nm JC-1 monomers** 505 nm 511 m BODIPY@ FL

C

5 -ceramide 505 un 512 nm BODIPY@ FL

C

5 sphingomyelin 489 nm 520 urn acridine orange 504 nm 511 nm LysoTrackerTM Green DND-26 508 nm (none) FUN-1TM 532 nm 545 nm LysoTrackerTM Green Br 2 534 nm 551 nm LysoTrackerTM Yellow DND 68 541 un 640 nm Neutral Red 528 nm 551 m rhodamine 6G 524 un 550 nm Tetrabromor hodamine 123 528 nrn 551 rn rhodamine 6G 533 nrn 545 un BODIPY@ FL Br 2 C 5 ceramide 546 un 590 un ethidium bromide 549 nm 565 nm DilC 18 (3) 549 un 565 nm DilC 16 (3) WO 00/79274 PCTUSOO/17380 45 485-585 590 nm JC-1 nm aggregates** 559 nm 568 nm Brefeldin A, BODIPY FL 558/568 conjugate isomer 1 Group III 425 - 440 450-535 Suitable for Use with Any Group III nm nm Donor: CFP-F64L/ 433 nm 475 nm 461 nm 585 nm 2-Di-1-ASP S65T/Y66W/ 501 nm* N1461/M153T/ V163A/N212L 461 nm 589 nm DASPEI 466 nm 536 nm NBD

C

6 ceramide 466 nm 536 nm NBD

C

6 sphingomyelin 483 nm (none) SYTO@ 18 484 nm 500 nm DiOC 5 (3) 484 nm 501 nm DiOC 6 (3) 485-585 590 nm JC-1 nm aggregates** 489 nm 520 nm acridine orange 502 nm 512 nm YFP S65G/Y66W/ S72A/T203Y 503 nm 510 nm Brefeldin A, BODIPY@ FL conjugate isomer 1 WO 00/79274 PCTUSOO/17380 46 504 nm 511 nm LysoSensorTM Green DND-26 505 nm 511 nm BODIPY@ FL

C

5 -ceramide 505 nm 512 nm BODIPY@ FL

C

5 sphingomyelin 508 nm (none) FUN-l TM 528 nm 551 nm rhodamine 6G 532 nm 545 nm LysoSensor TM Green Br, 534 nm 551 nm LysoTracker TM Yellow DND 68 541 nm 640 nm Neutral red Group IV 470 - 500 505 - 565 Suitable for Use with Any Group nm nm Donor: IV RFP-S65C 479 nm 507 nm 507 nm 529 nm rhodamine 123 RFP- 488 nm 507 nm 510 nm 527 nm JC-1 F64L/S65T monomers** RFP-S65T 489 nm 511 nm 524 nm 550 nm tetrabromorhod amine 123 MitoFluor TM 489 nm 517 nm 528 nm 551 nm rhodamine 6G Green RFP-F64M/ 490 nm 509 nm 548 nm 573 nm TMRM S65G/Q69L MitoTracker@ 490 nm 516 nm 549 nm 574 nm TMRE Green FM NAO 495 nm 519 nm 550 nm 574 nm tetramethylrosa mine wildtype 470 nm* 510 nm 556 nm 578 nm rhodamine B

GFP

WO 00/79274 PCT/USOO/17380 47 acridine orange 489 nm 520 nm 505 nm 511 nm BODIPY@ FL

C

5 -ceramide 505 nm 512 nm BODIPY@ FL

C

5 sphingomyelin 508 nm (none) FUN-I TM 533 nm 545 nm BODIPY@ FL Br 2

C

5 ceramide 534 nm 551 nm LysoTracker TM Yellow DND 68 541 nm 640 nm Neutral red 549 nm 565 nm DilC 18 (3) 549 nm 565 nm DilC 16 (3) 559 nm 568nm Brefeldin A, BODIPY FL 558/568 conjugate isomer I Group V 495 - 509 511 - 570 Suitable for Use with Any Group V nm nm Donor: YFP-S65G/ 502 nm 512 nm 510 nm 527 nm JC-1 Y66W/S72A/ monomers** T203Y 524 nm 550 nm tetrabromorhod amine 123 "FLASH" 508 nm 528 nm 528 nm 551 nm rhodamine 6G proteins 533 nm 545 nm BODIPY@ FL Br 2 C 5 ceramide 534 nm 551 nm LysoTrackerTM Yellow DND 68 WO 00/79274 PCTIUSOO/17380 48 541 nm 640 nm Neutral red 548 nm 573 nm TMRM 549 nm 574 nm TMRE 549 nm 565 nm DilC 18 (3) 549 nm 565 nm DilC 1 6 (3) 550 nm 574 nm tetramethylrosa mine 556 nm 578 nm rhodamine B 556 nm 585 nm 4 dimethylamino tetramethyl rosamine 559 nm 568 nm Brefeldin A, BODIPY FL 558/568 conjugate isomer I Group VI 545 - 560 565 - 625 Suitable for Use with Any Group nm nm Donor: VI MitoTracker@ 551 576 579 nm 601 nm DiOC 2 (5) Orange CMTMRos 589 nm 617 nm BODIPY@ TR ceramide * Minor excitation or emission peak. ** JC-1 monomers vs. JC-1 aggregates: at higher concentrations (aqueous solutions > 0.1 uM) or in mitochondria with higher potentials, and the "J-aggregates: have different spectral properties than the parent compound. 5 A variety of small, hydrophilic molecules can serve as ET donor and ET acceptor molecules. Such compounds can be used when it is desired to have a donor and/or acceptor compound undergo energy transfer in a water-based subcellular site or compartment. It may be desired in some aspects of the invention to have such WO 00/79274 PCT/USOO/17380 49 compounds preferentially accumulate in a water-based subcellular site or compartment. Some such compounds are known to preferentially accumulate at particular subcellular locations. Additionally or alternatively, a moiety that directs a compound to a subcellular location can be conjugated to a donor or acceptor moiety in order to 5 generate a donor or acceptor compound capable of preferentially accumulating at the subcellular location of choice. For example, published PCT application WO 98/17826, herein incorporated by reference, describes methods for conjugating mitochondria directing moieties to various compounds. Small lipophilic molecules, can be used when it is desired to have a 10 donor and/or acceptor compound preferentially accumulate in a cellular membrane, such membranes typically consisting in significant part of lipid bi-layers. Additionally or alternatively, a lipid or lipophilic molecule can be conjugated to a donor or acceptor moiety in order to generate a donor or acceptor compound capable of preferentially accumulating in a cellular membrane. 15 Examples of proteins that can serve as donor and acceptor compounds include fusion proteins comprising a "FLASH" (fluorescein arsenical helix binder) sequence (Griffin et al., Science 281:269-272, 1998), or an aequorin protein or a green fluorescent protein (Kendall et al., Trends in Biotechnology 16:216-224, 1998, and references cited therein). As used herein, the term "green fluorescent protein" 20 encompasses the wildtype green fluorescent protein (wildtype GFP), as well as blue shifted, cyan-shifted, red-shifted and yellow-shifted derivatives of wildtype GFP (designated, respectively, BFP, CFP, RFP and YFP; see published PCT application WO 98/06737). Table 2 includes descriptions of the amino acid changes in various green fluorescent protein derivatives and the respective excitation and emission peak 25 wavelengths of these GFP derivatives. In order to generate an expression construct that produces an aequorin, GFP or FLASH fusion protein that accumulates in the organelle or other subcellular site of interest, an expression vector comprising nucleotide sequences appropriate for gene expression can be manipulated to comprise (1) a first nucleic acid encoding a GFP 30 derivative or FLASH polypeptide and (2) a second nucleic acid encoding a peptide WO 00/79274 PCT/USOO/17380 50 sequence that directs a protein to an organelle or other subcellular site of interest (i.e., the "targeting sequence"), wherein the first and second nucleic acids are linked so as to have a common reading frame that comprises both nucleic acids. Such fusion proteins can be directed to a particular membrane within a cell (such as, for example, the nuclear 5 membrane or the inner or outer membrane of organelles such as mitochondria and chloroplasts), or to other specific subcellular locations, depending on the nature of the particular targeting sequence that is used in a given instance. Table 3 lists some non limiting examples of intracellular sites wherein the donor and acceptor compounds listed in Table 2 accumulate. 10 Table 3: Sites of Localization of Non-Protein Donor and Acceptor Compounds to Subcellular Compartments Subcellular Compartment Compounds Endoplasmic reticulum & BODIPY@ TR ceramide; DiOC 5 (3); NBD C 6 -ceramide; Golgi apparatus NBD C 6 -sphingomyelin; Brefeldin A; BODIPY@ FL conjugate isomer 1; BODIPY@ FL Cs-ceramide; BODIPY@ FL C 5 - sphingomyelin; BODIPY@ FL Br 2

C

5 ceramide; DilC I8(3); and DilC 1 6 (3) Lysosomes & other acidic acridine orange; FUN- 1 TM; hydroxystilbamidine, methane organelles sulfonate; LysoTrackerTMs Blue DND-22, Green Br 2 , Green DND-26, and Yellow DND-68; neutral red; LysoSensor

TM

s Blue DND-167, Blue DND-192, and Green DND-253 Mitochondria 2-Di-1-ASP; 4-Di-1-ASP; DASPEI; SYTO@ 18; DiOC 6 (3); rhodamine 123; tetrabromorhodamine 123; JC 1; ethidium bromide; rhodamine 6G; TMRM; TMRE; tetramethylrosamine; rhodamine B; 4-dimethylamino tetramethylrosamine; rhodamine 6G; DiOC 2 (5); also DiOC 7 (3) (plant mitochondria). A further criterion is that the donor and acceptor compounds should 15 accumulate in the subcellular compartment at the same site, which will permit ET to take place, or at acceptably adjacent sites. By "acceptably adjacent" it is meant that WO 00/79274 PCTUSOO/17380 51 such sites are within close enough proximity for ET to occur. Such sites are from about 100 Angstroms (A) to about 10 A or less from each other, typically about 80 A, 60 A, 50 A, 40 A, 30 A, 25 A, 20 A, 15 A, 10 A, 5 A or less from each other, preferably 70 A or less from each other, more preferably 50 A or less from each other, and most 5 preferably 40 A or less from each other, depending on the donor-acceptor pair of compounds. In any event, because the relationship of (i) the distance between an ET donor molecule and an ET donor molecule to (ii) the ability for ET to transpire is well established (see, e.g., Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes, Eugene, OR). those familiar with the art will 10 readily appreciate that donor-acceptor intermolecular distance is a cardinal determinative factor for the efficiency of ET. As a non-limiting example, one subcellular site of interest is the organelle known as the mitochondrion. The mitochondrion comprises an outer membrane that is exposed to the cytoplasm and with which various cytoplasmic factors 15 may transiently or stably associate, an inner membrane, an intermembrane space between the inner and outer membranes, and a matrix (the compartment within the inner membrane), arranged as is shown in Figure 1. For mitochondria, acceptably adjacent sites include (i) the outer membrane and the cytoplasm, including cytoplasmic factors associated with the outer membrane; (ii) the outer membrane and the intermembrane 20 space; (iii) the intermembrane space and the inner membrane; and (iv) the inner membrane and the matrix, including factors within the matrix. In the case of mitochondria, by way of example and not limitation, GFP fusion protein derivatives have been targeted to the mitochondrial matrix using cytochrome c oxidase subunit IV protein sequences (Llopis et al., Proc. Natl. Acad Sci. 25 U.S.A. 95:6803-6808, 1993), to the intermembrane space using cytochrome c protein sequences (Mahajan et al., Nature Biotech. 16:547-552, 1998), and to the outer membrane of mitochondria using hexokinase (Sui et al., Arch. Biochem. Biophys. 345:111-125, 1997), Bcl-2 or Bax (Mahajan et al., Nature Biotech. 16:547-552, 1998) protein sequences. GFP fusion proteins have also been targeted to mitochondria using 30 3-oxoacyl-CoA thiolase (Zhang et al., Biochem. Biophys. Res. Commun. 242:390-395, WO 00/79274 PCT/USOO/1 7380 52 1998), OSCP (Prescott et al., FEBS Letts. 411:97-101, 1997) and BNIP3 (Yasuda et al., J. Biol. Chem. 273:12415-12421, 1998) protein sequences. Aequorin fusion protein derivatives have been targeted to mitochondria using cytochrome c oxidase protein sequences (Pinton et al., Biofactors 8:243-253, 1998; Rizzuto et al., Nature 358:325 5 327, 1992). Other fusion proteins have been described that target mitochondrial sites using protein sequences from mitochondrial (or bacterial) thiolases (Arakawa et al., J. Biochem., Tokyo, 107:160-164, 1990), FO-ATPase subunit 9 (J. Biol. Chem. 271:25208 25212, 1996), manganese superoxide dismutase (Balzan et al., Proc. Natl. Acad Sci. US.A. 92:4219-4223, 1995), and P-450(SCC) (Kumamoto et al., J. Biochem., Tokyo, 10 105:72-78, 1989). In the case of chloroplasts, by way of example and not limitation, fusion proteins have been targeted to the outer membrane by use of the SCE70 heat shock protein targeting sequence (Wu et al., J Biol. Chem. 268:19384-19391, 1993). Other targeting sequences, such as those from the Rieske iron-sulfiur protein (Madueno et al., 15 J. Biol. Chem. 269:17458-17463, 1994), direct fusion proteins across the thylakoid membrane. If dual targeting to mitochondria and chloroplasts is desired, some fusion proteins comprising dual targeting sequences have been described (Creissen et al., Plant J. 8:167-175, 1995; Huang et al., Plant Cell 2:1249-1260, 1990). Conversely, when 20 plant cells are being used and targeting to only mitochondria or chloroplasts is desired, care must be taken to ensure that a dual targeting sequence is not employed. In the case of the nucleus, by way of example and not limitation, aequorin fusion protein derivatives have been targeted to the nucleus using nucleoplasmin protein sequences (Badminton et al., J. Biol. Chem. 271:31210-31214, 25 1997). In the case of the endoplasmic reticulum (ER), by way of example and not limitation, aequorin fusion protein derivatives have been targeted to the endoplasmic reticulum using calreticulin protein sequences (Kendall et al., Biochem. Biophys. Res. Commun. 189:1008-1016, 1992).

WO 00/79274 PCT/USOO/1 7380 53 In the case of the Golgi apparatus, by way of example and not limitation, aequorin fusion protein derivatives have been targeted to the Golgi plasma membrane using galactosyltransferase, SNAP-25, connexin and 5-HTIA-receptor protein sequences (Burton et al., Mol. Cell. Biol. 7:419-434, 1996; Marsault et al., EMBO J 16:1575 5 1581, 1997; Daguzan et al., Int. J. Dev. Biol. 39:653-657, 1995). GFP fusion proteins have been targeted to the Golgi apparatus using galactosyltransferase protein sequences (Llopis et al., Proc. Natl. Acad. Sci. US.A. 95:6803-6808, 1993) In the case of whole cell assays, another criterion is that the accumulation of ET donor and acceptor molecules should occur preferentially at sites 10 within the mitochondrion or whichever organelle or subcellular compartment is of interest. However, some accumulation of the compounds in other, secondary intracellular sites in acceptable, particularly if the donor and acceptor do not accumulate at the same secondary intracellular site (i.e., so that ET cannot occur in the secondary sites), or if the amount of background ET-derived signal is low enough that events 15 specific to the organelle of interest can be followed despite accumulation(s) of compound(s) at secondary sites. Moreover, most if not all of the assays described herein can be adapted for use with isolated organelles, in which instance preferential accumulation is not a criterion. Instrumentation for Detecting Energy Transfer 20 A variety of instruments can be used in methods of the invention to excite a donor compound and to measure emission from an acceptor compound. Which instrument(s) is (are) applicable for a particular donor-acceptor pair depends on factors such as (1) the need to apply energy at a wavelength that will excite the donor compound, preferably at or near kD(ex), to samples; (2) the need to measure energy 25 within the emission spectrum of the acceptor compound, preferably at or near XA(em); (3) the type of samples to be assayed in a given program; and (4) the number of samples to be assayed in a given program. With regard to factors (1) and (2), the spectra of energy being applied to samples to excite a donor compound, and the spectra of energy being emitted by an WO 00/79274 PCT/USOO/17380 54 excited acceptor compound and measured in samples will determine, in general, what type of instrument will be used. For example, although XD(em) should not be identical to XA(em), the minimal acceptable amount of difference between these two values will be influenced by, among other factors, the instrumentation being used. That is, as 5 XD(em) approaches XA(em), instruments capable of resolving closely-spaced wavelengths are required, and an assay using a donor-acceptor pair wherein the difference between XD(em) and XA(em) is less than about 3 to about 5 nm requires a high resolution instrument. Conversely, an assay using a donor-acceptor pair wherein the difference between XD(em) and kA(em) is greater than about 50 to about 75 nm 10 requires an instrument of medium to low resolution. With specific regard to factor (2), the type of energy being emitted by an excited acceptor compound and measured in samples will determine, in general, what type of instrument will be used. By definition, a fluorometer is a device that measures fluorescent energy and should therefor be part of the instrumentation. A fluorometer 15 may be anything from a relatively simple, manually operated instrument that accommodates only a few sample tubes at a time, to a somewhat more complex manually operated or robotic instrument that accommodates a larger number of samples in a format such as, e.g., a 96-well microplate (such as, e.g., anfmaxTM fluorimetric plate reader, Molecular Devices Corp., Sunnyvale, CA; or a Cytofluor fluorimetric plate 20 reader, model #2350., Millipore Corp., Bedford, MA), or a complex robotic instrument (such as, e.g., a FLIPRTM instrument; see infra) that accommodates a multitude of samples in a variety of formats such as 96-well microplates. With regard to factor (3), the type of samples to be assayed in a given program, different formats will be appropriate for different types of samples. For 25 example, 96-well microplates are suitable in instances where the cells or isolated organelles of interest adhere to the material of the microplate or to some material applied to the wells of the microplate; however, plastic fluorescence results in a larger background component at excitation wavelengths below about 400 nm. For measurements involving nonadherent cells or organelles, or soluble extracts prepared 30 therefrom, an instrument capable of reading fluorescent signals in glass or polymeric WO 00/79274 PCT/USO0/17380 55 tubes or tubing is preferred. Regardless of what type of format is used, it should allow for the introduction of donor and acceptor compounds, as well as control reagents and compounds being evaluated, into the samples at appropriate points in time. Factor (4), the number of samples to be assayed in a given program, will 5 influence how automated the instrument will be. For example, when high throughput (HTS) assaying of a large number of samples is desired, robotic or semi-robotic instruments are preferred. However, a fair number of samples can be processed manually, particularly when formats that accommodate large sample numbers (such as, e.g., 96-well microplates) are used. 10 Depending on the assay, a Fluorometric Imaging Plate Reader (FLIPRTM) instrument (Molecular Devices, Sunnyvale, CA) is often the instrument of choice for ET-based assays of the invention. The FLIPRM system (see http://www.moleculardevices.com/pages/flipr.html) has the following desirable features: it uses a combination of a water-cooled, argon-ion laser illumination and 15 cooled CCD camera as an integrating detector that accumulates signal over the period of time in which it is exposed to the image and, as a result, its signal-to-noise characteristics are generally superior to those of conventional imaging optics; it also makes use of a proprietary cell-layer isolation optics that allow signal discrimination on a cell monolayer, thus reducing undesirable extracellular background fluorescence; it 20 provides data in real-time, and can also provide kinetic data (i.e., readings at a multitude of timepoints); it has the ability to simultaneously stimulate and read all 96 wells of a 96-well microplate; it provides for precise control of temperature and humidity of samples during analysis; it includes an integrated state-of-the-art 96-well pipettor, which uses dispensable tips to eliminate carryover between experiments, that can be 25 used to aspirate, dispense and mix precise volumes of fluids from microplates; and, in the case of the FLIPR384 instrument, it can be adapted to run sample assays in a robotic or semi-robotic fashion, thus providing for analysis of large numbers of samples in shortest amount of time (e.g., up to about a hundred 96-well microplates per day).

WO 00/79274 PCT/USOO/17380 56 Monitoring Conditions or Processes within Subcellular Compartments The term "subcellular compartment" refers to any intracellular space that is, for at least some of the time, maintained in an at least partially isolated condition. Some type of physical barrier, typically a bilipid membrane, forms the border between a 5 given subcellular compartment and other cellular components. A border around a subcellular compartment may be permeable, impermeable, or semi-permeable to molecules inside or outside the subcellular compartment. Subcellular compartments include, but are not limited to, known organelles such as, e.g., in a eukaryotic cell, the nucleus, the nucleolus, mitochondria, chloroplasts, endosomes, lysosomes, endoplasmic 10 reticulum, Golgi apparatus, and the like. The present invention can also be used with extracellular subcellular structures that interact with and/or are internalized by cells including, by way of example and not limitation, viruses and other intracellular parasites. Some of the subcellular compartments that can be monitored or assayed using the present invention, and applications particular for each such subcellular 15 compartment, are described in more detail in the following subsections. Mitochondria One subcellular compartment of particular interest is the organelle known as the mitochondrion (plural, mitochondria). Mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical 20 regulation of a wide array of cellular respiratory, oxidative and metabolic processes. These include electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis. 25 In addition to their role in energy production in growing cells, mitochondria (or, at least, mitochondrial components) participate in programmed cell death (PCD), also known as apoptosis (Newmeyer et al., 1994, Cell 79:353-364; Liu et al., 1996, Cell 86:147-157). Apoptosis is apparently required for normal development of the nervous system and functioning of the immune system. Moreover, some disease WO 00/79274 PCT/USOO/17380 57 states are thought to be associated with either insufficient or excessive levels of apoptosis (e.g., cancer and autoimmune diseases in the first instance, and stroke damage and neurodegeneration in Alzheimer's disease in the latter case). For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed (1998, Science 5 281:1309-1312), Green (1998, Cell 94:695-698) and Kromer (1997, Nature Medicine 3:614-620). Thus, agents that affect apoptotic events, including those associated with mitochondrial components, might have a variety of remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive uses. A variety of apoptogens are known to those familiar with the art (see, 10 e.g., Green et al., 1998 Science 281:1309 and references cited therein) and may include by way of illustration and not limitation: tumor necrosis factor-alpha (TNF-ca); Fas ligand; glutamate; N-methyl-D-aspartate (NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et al., 1997 J. Cell. Biol. 138:449-469); paraquat (Costantini et al., 1995 Toxicology 99:1-2); ethylene glycols; protein kinase inhibitors, such as, e.g., 15 staurosporine, calphostin C, caffeic acid phenethyl ester, chelerythrine chloride, genistein; 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; N-[2-((p bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide; KN-93; quercitin; d erythro-sphingosine derivatives; UV irradiation; ionophores such as, e.g.: ionomycin and valinomycin; MAP kinase inducers such as, e.g.: anisomycin, anandamine; cell 20 cycle blockers such as, e.g.: aphidicolin, colcemid, 5-fluorouracil, homoharringtonine; acetylcholinesterase inhibitors such as, e.g., berberine; anti-estrogens such as, e.g.: tamoxifen; pro-oxidants, such as, e.g.,: tert-butyl peroxide, hydrogen peroxide; free radicals such as, e.g., nitric oxide; inorganic metal ions, such as, e.g., cadmium; DNA synthesis inhibitors such as, e.g.: actinomycin D; DNA intercalators such as, e.g., 25 doxorubicin, bleomycin sulfate, hydroxyurea, methotrexate, mitomycin C, camptothecin, daunorubicin; protein synthesis inhibitors such as, e.g., cycloheximide, puromycin, rapamycin; agents that affect microtubulin formation or stability such as, e.g.: vinblastine, vincristine, colchicine, 4-hydroxyphenylretinamide, paclitaxel; Bad protein, Bid protein and Bax protein (see, e.g., Jurgenmeier et al., 1998 Proc. Nat. Acad WO 00/79274 PCT/USOO/17380 58 Sci. USA 95:4997-5002 and references cited therein); calcium and inorganic phosphate (Kroemer et al., 1998 Ann. Rev. Physiol. 60:619). Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the 5 cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes (see Figure 2). The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix. (For a review, see, e.g., Emster et al., 1981 J. Cell Biol. 91:227s.) The cristae, originally 10 postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube like conduits that may form networks, and that can be connected to the inner membrane by open, circular (30 nm diameter) junctions (Perkins et al., 1997, Journal of Structural Biology 119:260). While the outer membrane is freely permeable to ionic and non-ionic 15 solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (> ~10 kDa) molecules. Four of the five multisubunit protein complexes (Complexes I, III, IV 20 and V) that mediate ETC activity are localized to the inner mitochondrial membrane. The remaining ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place within the ETC, protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical potential of 25 approximately 220 mV referred to as the "protonmotive force" (PMF), which is often represented by the notation AT or A'm. APm represents the sum of the electric potential and the pH potential (i.e., the pH differential) across the inner mitochondrial membrane (see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s and references cited therein).

WO 00/79274 PCT/USOO/17380 59 ATm provides the energy for phosphorylation of adenosine diphosphate (ADP) to yield ATP by ETC Complex V, a process that is coupled stoichiometrically with transport of a proton into the matrix. ATm is also the driving force for the influx of cytosolic Ca 2 + into the mitochondrion. Under normal metabolic conditions, the inner 5 membrane is impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the sole means whereby protons can return to the matrix. When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during mitochondrial permeability transition (MPT) that accompanies certain diseases associated with altered mitochondrial function, protons 10 are able to bypass the conduit of Complex V without generating ATP, thereby uncoupling respiration from energy production. During MPT, ATm collapses and mitochondrial membranes lose the ability to maintain an equilibrium distribution of one or more ionic species or other solutes, i.e., to selectively regulate permeability to solutes small (e.g., ionic Ca 2 ", Na*, K*, H') and/or large (e.g., proteins). 15 Loss of mitochondrial membrane electrochemical potential may be the result of mechanisms such as free radical oxidation, or may be due to direct or indirect effects of mitochondrial and/or extramitochondrial gene products. Loss of mitochondrial potential appears to be a critical event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases such as 20 Alzheimer's Disease; diabetes mellitus; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome. To provide improved therapies for such 25 diseases, agents that limit or prevent loss of mitochondrial membrane potential (Ay.) may be beneficial. The present invention provides a novel approach to the identification of agents useful for such diseases. The invention fulfills the need for an assay that permits rapid screening for agents capable of altering mitochondrial membrane potential and provides other related advantages.

WO 00/79274 PCT/USOO/17380 60 Assays for Measuring Changes in Parameters in Subcellular Compartments When the ET-based assay is designed to measure a change in state of, or decrease or increase in some activity at, a subcellular compartment or site, such as Ay of mitochondria or the presence or absence of factors that are transiently associated with 5 or released from an intracellular site, an additional criterion for donor-acceptor compounds is that one of the compounds (either the donor or the acceptor compound) must accumulate in and/or be released from the subcellular compartment or site in a manner that is dependent on the chosen parameter or activity, whereas the presence of the other compound (the acceptor or donor, respectively) in the subcellular 10 compartment must be independent of the chosen parameter or activity. Compounds whose mitochondrial concentration is dependent on Ay include, by way of example and not limitation, TMRM (Farkas et al., Biophys. J. 56:1053-1069, 1989), TMRE (Ehrenberg et al., Biophys. J. 53:785-794, 1988), rhodamine 123 (Scaduto et al., Biophys. J. 76:469-477, 1999), ethidium bromide 15 (Coppey-Moisan et al., Biophys. J. 71:2319-2328, 1996), DASPMI (4-Di-1-ASP and 2 Di-1-ASP) and DASPEI (Rafael et al., FEBS Lett. 170:181-185, 1984). Compounds whose mitochondrial concentration is not dependent on Ay include, by way of example and not limitation, NAO (Maftah et al., Biophys. Res. Commun. 164:185-190, 1989), MitoTracker@ Green FM and MitoFluorTM Green (Haugland, Handbook of Fluorescent 20 Probes and Research Chemicals, 6th Ed., Molecular Probes, Inc., Eugene, OR, 1996, p. 269), and DAPI (Coppey-Moisan et al., Biophys. J. 71:2319-2328, 1996). Both collapse and dissipation of Ay can be monitored using such compounds. As used herein, "Ay collapse" refers to the rapid dissolution of Ay, i.e., Ay reaches zero within a few minutes after mitochondria are treated with an agent that induces collapse of 25 mitochondrial membrane potential, such as, for instance CCCP or FCCP or any other agent capable of rapidly driving Aym to zero. The term "Ay dissipation" refers to a slower decrease in Ay that does not result in Ay reaching zero within a few minutes (although this may happen over a longer time frame or after repeated exposures) after mitochondria are treated with an agent that induces dissipation of mitochondrial 30 membrane potential, such as, for example, ionomycin, thapsigargin, atractyloside, WO 00/79274 PCT/USOO/17380 61 A23187, 4-bromo-A23187, adenine nucleotide translocator inhibitors, inhibitors of mitochondrial electron transport chain (ETC) complex I, inhibitors of ETC complex II in the presence of a complex I substrate, other partial inhibitors of the ETC or other agents that lead to an increased intramitochondrial calcium concentration as a result of 5 elevated intracellular cytosolic free calcium concentration. Those having ordinary skill in the art are familiar with any number of mitochondrial ETC inhibitors that have been characterized with regard to which ETC components may be impaired. For additional disclosure relating to measurement of mitochondrial membrane potential, agents that induce collapse of mitochondrial membrane potential and agents that induce dissipation 10 of mitochondrial membrane potential, see U.S. Application Serial Nos. 09/161,172 and 09/185,904. Using mitochondria as an example, a variety of factors are known to be either (1) transiently associated with the outer membrane of the mitochondrion or (2) typically located at an intramitochondrial site but released from mitochondria during 15 events such as, e.g.. mitochondrial pore transition (MPT) or apoptosis (a.k.a. programmed cell death, PCD; for a review, see Green et al., Science 281:1309-1312, 1998). Examples of proteins belonging to class (1) include hexokinase II, and Bcl-2, Bcl-XL, Bax and other members of the bcl-2 gene family (Kroemer, Nature Med 3:614 620, 1997; Nartita et al., Proc. Natl. Acad Sci. U.S.A. 95:14681-14686, 1998). 20 Examples of class (2) factors that are released during MPT or apoptosis include cytochrome c (Yang et al., Science 275:1129-1132, 1997; Kluck et al., Science 275:1132-1136, 1997), procaspase-2 and -9 (Susin et al., J. Exp. Med 189:381-394, 1998) and apoptosis inducing factor (AIF; Susin et al., . Exp. Med 184:1331-1341, 1996; Susin et al., J. Exp. Med 186:25-37, 1997). Nucleic acids comprising nucleotide 25 sequences that encode these proteins can be used to construct fusion proteins with FLASH, aequorin or green fluorescent proteins such as wildtype GFP, BFP, CFP, RFP and YFP in order to construct fluorescent derivatives that exhibit the same transient associations with mitochondria, or releases from mitochondria, as the corresponding parent proteins. For example, hexokinase II fusion proteins that associate with the outer 30 membrane of mitochondria (Sui et al., Arch. Biochem. Biophys. 345:111-125, 1997), WO 00/79274 PCT/USOO/17380 62 and cytochrome c fusion proteins that localize GFP (Mahajan et al., Nature Biotech. 16:547-552, 1998) or other proteins (Nye et al., Mol. Cell. Biol. 10:5763-5771, 1990) to the intermembrane space of mitochondria, have been described. FLASH, aequorin and green fluorescent fusion proteins are used as donor or acceptor compounds in FRET 5 based assays designed to monitor the degree and/or rate of mitochondrial association or release of factors having various biological functions. The ET-based methods of the invention possess certain advantages over other methods for assaying Aym. For example, methods that utilize a single potentiometric fluorophore (i.e., a fluorophore that accumulates in mitochondria in a 10 Apm-dependent manner) may require that the fluorophores be present at concentrations that are toxic when agents that impact Aym are introduced (see, e.g., U.S. Patent No. 5,169,788). In contrast, the ET-based assays of Aym of the invention can be carried out using lower, non-toxic doses of fluorophores. Furthermore, plasma membrane potential contributes to the signal in assays where a single potentiometric fluorophore is used, 15 whereas the ET-based assays of the invention are specific for changes in mitochondrial membrane potential. The detected fluorescence emission is typically compared to a reference signal. For quantitative measurements of ATm, the reference signal may be the signal observed in mitochondria with a known ATm, and one or more such references signals 20 may be used. Alternatively, ATim may be evaluated relative to a ATm within the same type of mitochondria (e.g., mitochondria derived from the same subject or biological source), under certain specific conditions, to evaluate changes in ATm, or relative to a ATm in a different type of mitochondria (e.g., mitochondria derived from a distinct subject or biological source). Specific embodiments of the present invention may 25 employ different reference signals, as described in more detail below. Chloroplasts The chloroplast is an organelle found in plant cells wherein photosynthesis takes place. Photosynthesis, in addition to being an integral part of a plant cell's metabolism, is an important process that impacts many other living WO 00/79274 PCTUSOO/17380 63 organisms as well. The reason for this is twofold: photosynthesis "fixes" atmospheric

CO

2 into biologically usable carbohydrate (CHO), molecules and also produces 02 which is required by all aerobic organisms. Like mitochondria, chloroplasts have a double (outer and inner) 5 membrane, contain their own DNA and have translation factors (ribosomes, tRNAs, etc.) that are distinct from those found in the cytoplasm. Electron microscopy demonstrates that, like mitochondria, chloroplasts have a highly organized internal ultrastructure which includes flattened membranous bodies known as lamellae or thykaloid discs. Chloroplasts are, however, typically much larger than mitochondria; in 10 higher plants they are generally cylindrical in shape and range from about 5 to 10 p1 in length and from 0.5 to 2 p in diameter. Like mitochondria, which are present in greater numbers in certain tissues (e.g., liver) than others, chloroplasts have greater copy numbers in some tissues than others. For example, mature leaves contain many chloroplasts and the total amount of chloroplast DNA in such leaves is about twice that 15 of nuclear DNA (Jope et al., J Cell. Biol. 79:631-636, 1978). The Nucleus and the Nucleolus The nucleus is the organelle that comprises most (from the standpoint of information, if not mass) of a cell's DNA in the form of several chromosomes (Mitochondria and chloroplasts have their own DNA molecules that are typically much 20 smaller than the nuclear genomes, and thus encode fewer functions; however, as a cell contains only one nucleus and may contain many mitochondria and/or chloroplasts, the total mass of the DNA molecules in these organelles may approach that of the nuclear DNA.) The nucleus is bounded by two membranes collectively called the nuclear envelope (the membranes are known as the inner and outer nuclear membranes). 25 Macromolecules, most particularly RNA molecules, are conveyed to or from the cytosol through openings in the nuclear envelope called nuclear pores. The nucleolus is a subcompartment of the nucleus. In contrast to the remainder of the nucleus, wherein messenger (mRNA) molecules are transcribed from WO 00/79274 PCT/USOO/17380 64 DNA, it appears that it is mainly ribosomal RNA (rRNA) molecules that are produced in the nucleolus. Endosomes, Lysosomes and Peroxisomes Cells assimilate extracellular fluid, and macromolecules dissolved 5 therein, by a process called endocytosis. Endocytotic vesicles are formed when a portion of the cell membrane evolves from a cup-shaped surface feature into an inwardly-directed "bud" and, eventually, a small membrane-bound vesicle that is taken up into the cytosol. At least two mechanisms have been proposed for the formation of the cup-shaped surface features from which endosomes originate. First, local changes 10 in the structure and/or composition of the lipid bilayer portion of the cell membrane can induce membrane curvature over a limited area thereof. Second, one or more coat proteins can act on a given location in the cell membrane to induce the formation of a cup-shaped surface feature. In the latter instance, the most well-characterized example are the "coated pits" that are formed, at least in part, by the protein clathrin (for a 15 review, see Schekamn and Orci, Science 271:1526-1533, 1996). Lysosomes contain various hydrolytic enzymes, each of which catalyzes the breakdown of specific types of macromolecules. Primary lysosomes containing such enzymes are produced intracellularly and may fuse with endosomes to form secondary lysosomes. In the latter type of vesicle, the enzymes from the primary 20 lysosome are brought into contact with, and are thus free to act upon, the contents of the endosome. In general, after enzymatic digestion of the contents of the secondary lysosome, its membrane is dissolved in order to release its contents into the cytosol. The formation and fate of, e.g., secondary lysosomes can be followed using the methods of the invention in the following manner. Cells are engineered to 25 produce one or more lysosomal enzymes modified to contain a moiety capable of serving as an acceptor or donor in energy transfer. Such cells are brought into contact with an agent that is taken up in endosomes, wherein the agent is or has been modified to be an ET acceptor or donor, respectively. When the resultant endosomes fuse with a primary lysosome, the acceptor and donor are present in the same subcellular WO 00/79274 PCT/USOO/17380 65 compartment (the secondary lysosome), and ET occurs and can be monitored as described herein. The dissolution of the secondary lysosome liberates the ET acceptor donor pair of molecules, which are then separated from each other as they are diluted into the cytosol, wherein the degree of ET decreases or ceases altogether. 5 Peroxisomes are another type of intracellular vesicles bounded by a single membrane. Unlike lysosomes, which generally contain hydrolytic enzymes, peroxisomes contain oxidative enzymes that generate and destroy hydrogen peroxide. Endoplasmic Reticulum The endoplasmic reticulum (ER) is composed of a series of flattened 10 sheets, tubes and sacs that enclose a large intracellular space. The membrane of the ER is in structural continuity with the outer nuclear membrane and extends throughout the cytoplasm. Some functions of the ER include the synthesis and transport of membrane proteins and lipids. Generally speaking, two types of ERs may exist in a cell. Smooth ER is generally tubular in shape and is typically devoid of attached ribosomes; one 15 major function of smooth ER is lipid metabolism. Rough ER typically occurs as flattened sheets, the cytosolic side of which is usually associated with many active (protein-synthesizing) ribosomes. Golgi Apparatus The Golgi apparatus is a system of stacked, flattened and membrane 20 enclosed sacs and is generally thought to be involved in the modification, sorting and packaging of macromolecules for secretion or for delivery to other subcellular compartments. Numerous small (> -50 nM) membrane-enclosed vesicles are thought to comprise macromolecules in order to carry out the transport thereof between the Golgi apparatus and other subcellular compartments. 25 Suborganellar Compartments Certain components of organelles are also subcellular compartments within the scope of the invention. For example, mitochondria, chloroplasts and nuclei WO 00/79274 PCT/US00/17380 66 are surrounded by two membranes. The space between a set of paired membranes is not itself an organelle, but is a subcellular compartment as defined herein. Such spaces are named, e.g., and respectively, the mitochondrial intermembrane space, the chloroplast intermembrane space, the nuclear intermembrane space, etc. Conditions and processes 5 within such spaces can be monitored according to the present invention by incorporating an acceptor-donor pair of molecules into the intermembrane, or by incorporating a donor or acceptor into the intermembrane space and an acceptor or donor, respectively, into either the inner or outer membrane. The subcellular compartment may also be a membrane per se. In this 10 aspect of the invention, membrane-directed donors [such as, e.g., 9-anthrylvinyl (LAPC)] and acceptors such as 3-perylenoyl (LPPC) are incorporated into one or more membranes of choice. The partition coefficients between membrane and aqueous phases are 8.3 x 105 and 10.5 x 105 for LAPC and LPPC, respectively (Razinkov et al., Biochim. Biophys. Acta 1329:149-158, 1997). 15 Intracellular Parasites Other subcellular compartments of interest include intracellular parasites such as viruses and intracellular bacteria such as Rickettsiae and Chlamydia spp. Viruses consist of a genome, which may be composed of either DNA or RNA, that is surrounded by a protein shell. In the case of animal viruses, this protein shell is often 20 itself enclosed within an envelope comprising both protein and lipid. Viruses multiply only within cells, as they are dependent on the host cells' macromolecular synthetic processes. They have thus been described as "genetic parasites." One example of how the present invention may be applied to such intracellular parasites, provided by way of illustration and not limitation, is as follows. 25 A viral particle typically consists of a "coat" or capsid surrounding one or more nucleic acids. The capsid, which typically comprises one or more structural polypeptides, protects the viral nucleic acids in extracellular environments, but must (if the viral nucleic acids are to be liberated and replicated) be removed after the virus is internalized by a host cell. The process by which the capsid is removed is called WO 00/79274 PCT/US0O/17380 67 "uncoating" and typically takes place in the cytoplasm (or a subcellular compartment, such as a vacuole, within the cytoplasm). Most animal viruses undergo uncoating as a result of the action of intracellular proteases on polypeptides that are a part of the capsid. Agents that inhibit or block viral uncoating, for example by inhibiting the 5 action of intracellular proteases, are expected to be novel antiviral agents; a method of assaying viral uncoating would be useful for screening for such agents. The present invention provides such a method for assaying viral uncoating in, for example, the following manner. Viral particles are prepared that contain an acceptor-donor pair of molecules ("loaded viruses"); this can be 10 accomplished by, e.g., contacting viral particles or cells infected with viruses with a donor-acceptor pair of molecules that specifically localize to lipid membranes. By way of example and not limitation, the donor can be 9-anthrylvinyl (LAPC) and the acceptor can be 3-perylenoyl (LPPC) (Razinkov et al., Biochim. Biophys. Acta 1329:149-158, 1997). 15 Viral adsorption typically occurs equally well at 4'C and 37 0 C, whereas uncoating proceeds rapidly at 37 0 C, but slowly, if at all at 4'C. Accordingly, loaded viruses are contacted with cells at 4'C for a period of time to allow for complete adsorption, after which the temperature is raised to 37*C to allow uncoating to proceed. As uncoating of the loaded viruses proceeds, the donor-acceptor molecules are released 20 from the capsid and they thus lose proximity to each other. This loss of proximity will be reflected in either an increase in fluorescence (if one molecule quenches the fluorescence of the other) or a decrease (if fluorescence is produced when the donor acceptor molecules are in close proximity to each other). The rate of change in fluorescence thus correlates with viral uncoating. When added to this assay system, an 25 agent that inhibits viral uncoating will reduce or eliminate the change in fluorescence. Rickettsia are small, pleiomorphic, gram-negative coccobacilli that have adapted to intracellular growth in arthropods and other organisms. Except for R. quintana (the agent of trench fever), all rickettsiae require living cells for growth. Species differ in terms of the location of intracellular multiplication; for example, R. 30 tsutsugamushi typically grow only in the cytoplasm, organisms of the spotted fever WO 00/79274 PCTIUSO0/17380 68 group grow both in the cytoplasm and the nucleus, and C. burnetii grows within the cytoplasm and phagolysosomes. Chlamydiaceae is a family of obligate intracellular bacterial parasites that infect a number of vertebrate hosts, typically birds or mammals (including 5 humans). The distinct developmental cycle of Chlamydia begins with the attachment to, and internalization by, a host cell by an elementary body (the metabolically dormant, extracellular phase of Chlamydia). Phagocytized elementary bodies develop into reticulate bodies that multiply by binary fission. Elementary body progeny are formed from the replicated reticulate bodies and released when the host cells rupture. 10 The life-cycle of Chlamydia presents another non-limiting example of how the invention may be applied to intracellular parasites. Chlamydia survive intracellularly within phagosomes, in part because the elementary body cell wall appears to inhibit fusion of the phagosomes with lysosomes that contain hydrolytic enzymes that would degrade the elementary bodies if phagolysosomes were formed. 15 When elementary bodies are labeled with a donor or acceptor molecule, and lysosomes with an acceptor or donor molecule, respectively, energy transfer will occur if phagolysosomes are formed. Agents that inhibit the elementary body's ability to prevent fusion of phagosomes and lysosomes will result in energy transfer that can be monitored by the present invention; such agents are expected to be novel antibiotics 20 useful for treating Chlamydia infections. Assaying Interactions Between Macromolecules within or Associated with Subcellular Compartments In another aspect of the invention, energy transfer is used to monitor interactions between pairs of macromolecules found within or associated with 25 subcellular compartments. This embodiment, which is drawn to means for monitoring the association of a macromolecular species and an organelle or other subcellular compartment, should not be confused with systems in which energy transfer in used to evaluate the interaction between two types of macromolecules.

WO 00/79274 PCTUSOO/17380 69 As one example, some cancer cells are thought to result, at least in part form overexpression of a protein that may preferentially associate with one or more subcellular compartments. The bcl-2 gene was initially identified as a causal factor in certain types of lymphatic cancers (B-cell lymphoma, hence the name) in which bcl-2 is 5 overexpressed, resulting in an abnormally longer lifespan for B-cells, which in turn is thought to allow these cells to accumulate additional mutations resulting in frank malignancy and lymphatic tumor development (for reviews of the Bcl-2 family of proteins, see Davies, Trends in Neuroscience 18:355-358, 1995; Kroemer, Nature Med. 3:614-620, 1997; W095/13292; W095/00160; and U.S. Pat. No. 5,015,568). 10 Although the biochemical function of Bcl-2 is not known (i.e., it is not clear whether it acts as an enzyme, receptor or signaling molecule), it is known to be localized to the outer mitochondrial membrane, the nuclear membrane and the endoplasmic reticulum. Another member of the Bcl-2 family of proteins, Bax, localizes to the outer mitochondrial membrane. Although FRET has been used to demonstrate 15 the interaction of Bcl-2 and Bax in individual mitochondria (Mahajan et al., Nat. Biotechnol. 16:547-552, 1998), energy transfer has not been used to monitor the association (or dissociation) of such proteins with (or from) subcellular compartments. The present invention provides methods for monitoring the interactions of macromolecules with subcellular compartments. 20 One example of such a method is as follows. The width of the combined inner and outer mitochondria membranes has been estimated to be 22 + 4 nm (Perkins et al., J. Structural Biol. 119:260-272, 1997). Accordingly, loading the intermembrane space with donor (or acceptor) molecules would be expected to bring them in sufficiently close proximity with acceptor (or donor) molecules present within or 25 associated with the outer mitochondrial membrane. Events such as the localization of Bcl-2 proteins to the outer mitochondrial membrane could thus be monitored by tagging Bcl-2 with an acceptor (or donor) that undergoes energy transfer with a donor (or acceptor) that has been loaded into the intermembrane space. In like fashion, the dissociation of proteins such as cytochrome c from mitochondria can be followed using 30 donor- or acceptor-tagged cytochrome c proteins and acceptor- or donor-loaded WO 00/79274 PCTUSOO/17380 70 (respectively) intramembrane spaces. Such processes are thought to represent significant events in apoptotic pathways (Green et al., Science 281:1309-1312, 1998; Green, Cell 94:695-698, 1998). Screening for Species-Specific Agents 5 In certain embodiments, the present invention provides screening assays for identifying species-specific agents. A "species-specific agent" refers to an agent that affects a subcellular compartment of a first organism belonging to one species but that does not affect the homologous subcellular compartment of a second organism belonging to another species. Thus the invention provides a method for identifying an 10 agent that preferentially alters a cellular membrane potential in a subcellular compartment of a first biological source without substantially altering a corresponding cellular membrane potential in a subcellular compartment of a second biological source. In preferred embodiments, the subcellular compartment is a mitochondrion and the cellular membrane potential is mitochondrial membrane potential. The screening 15 assays provided by the instant methods are thus directed in pertinent part to assaying, in the absence and presence of a candidate agent, a cellular membrane potential by contacting each of a first and second sample comprising one or more cellular membranes from a first and a second distinct biological source, respectively, with an ET donor and an ET acceptor molecule, exciting the ET donor to produce an excited ET 20 donor molecule, detecting a signal generated by energy transfer from the ET donor to the ET acceptor and comparing the signal generated in the absence of the candidate agent to the signal generated in the presence of the candidate agent. In those certain preferred embodiments wherein the invention is directed to a method for identifying an agent that preferentially alters mitochondrial membrane 25 potential in mitochondria from a first biological source without substantially altering mitochondrial membrane potential in mitochondria from a second biological source, neither the ET donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and the ET donor and the ET acceptor each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial WO 00/79274 PCT/USOO/17380 71 sites as provided herein. Typically, based upon the teachings provided herein, a person having ordinary skill in the art can readily determine when a candidate agent alters a cellular membrane potential such as mitochondrial membrane potential, for example, by detecting a statistically significant change in the membrane potential in the presence of 5 the agent relative to the potential detected in the absence of the agent. Methods for determining mitochondrial membrane potential are also provided in U.S. application number 09/161,172. As used herein, an agent identified according to the instant method that is a species-specific agent or an agent that "preferentially" alters mitochondrial 10 membrane potential in the mitochondria from a first biological source (e.g., a first species) without substantially altering the mitochondrial membrane potential in the mitochondria from a second biological source (e.g., a second species) refers to an agent that, following contact with mitochondria or cells of the first and second species, effects the continued viability of the mitochondria or cells from one of the species (i.e., either 15 the first or the second species but not both) while effecting the death or growth impairment of the mitochondria or cells from the other species. Similarly, where such an agent does not "substantially" alter mitochondrial membrane potential in the mitochondria of the first species refers to an agent that, following contact with mitochondria or cells of the first and second species, effects the continued viability of 20 the mitochondria or cells from one of the species (i.e., either the first or the second species but not both) while effecting the death or growth impairment of the mitochondria or cells from the other species. Thus, preferential alteration of mitochondrial membrane potential by such an agent may increase or may decrease AT., as long as the effect is species-specific. Without wishing to be bound by theory, cells 25 that undergo death or growth impairment in a species-specific manner as a result of contact with such an agent identified according to the instant method may do so by becoming apoptotic or necrotic, by entering cell cycle arrest or by becoming cytostatic, or by failing to remain viable or capable of growth by any other mechanism. In certain other embodiments an agent identified according to the instant 30 method that that "preferentially" alters mitochondrial membrane potential in the WO 00/79274 PCTUSOO/17380 72 mitochondria from a first biological sample (e.g., a first tissue) without substantially altering the mitochondrial membrane potential in the mitochondria from a second biological sample (e.g., a second tissue) refers to an agent that, following contact with mitochondria or cells of the first and second biological samples, effects the continued 5 viability of the mitochondria or cells from one of the samples (i.e., either the first or the second tissue samples but not both) while effecting the death or growth impairment of the mitochondria or cells from the other sample. Similarly, where such an agent does not "substantially" alter mitochondrial membrane potential in the mitochondria of the first sample refers to an agent that, following contact with mitochondria or cells of the 10 first and second species, effects the continued viability of the mitochondria or cells from one of the samples (i.e., either the first or the second samples but not both) while effecting the death or growth impairment of the mitochondria or cells from the other species. Thus, preferential alteration of mitochondrial membrane potential by such an agent may increase or may decrease AT, as long as the effect is sample-specific. 15 According to these embodiments, an agent may be identified that acts selectively in a tissue-specific manner, such that the agent may be employed to manipulate mitochondrial membrane potential in certain tissue types but not other, even within the same organism. Alternatively, the first and second tissues may be derived from distinct subjects of the same species, or from subjects of distinct species. For example, 20 according to such a method of the instant invention, an agent may be identified using this approach that preferentially alters neuronal cell mitochondrial membrane potential without substantially altering liver cell mitochondrial membrane potential. Using mitochondria as an example of a subcellular compartment, this embodiment of the invention may be used, for example, to identify agents that 25 selectively induce collapse of Ay in mitochondria derived from different species, e.g., in trypanasomes (Ashkenazi et al., Science 281:1305-1308, 1998), and other eukaryotic pathogens and parasites, including but not limited to insects, but which do not induce Ay collapse in the mitochondria found in the cells of their mammalian hosts. Such agents are expected to be useful for the prophylactic or therapeutic management of such 30 pathogens and parasites.

WO 00/79274 PCT/USOO/17380 73 For example, members of the phylum Apicomplexa (formerly called Sporozoa) comprise a large and diverse group of pathogenic protozoa that are intracellular parasites. Some members, including species of Babesia, Theileria and Eimeria, cause economically important animal diseases, and other members, such as 5 Toxoplasma gondii and Cryptosporidium spp. also cause human disease, particularly in immunocompromised individuals. The acomplexicans are unusual in terms of their extrachromosomal DNA elements, as they comprise both a mitochondrial genome and a putative plastid genome (see Feagin, Annu. Rev. Microbiol. 48:81-104, 1994, for a review). Probably the most well-studied acomplexicans are species of Plasmodium, 10 which cause malaria. Antimalarial agents include agents that specifically impact the function of Plasmodium mitochondria (Peters et al., Ann. Trop. Med. Parsitol. 78:567 579, 1984; Basco et al., J. Eukaryot. Microbiol. 41:179-183, 1994), and one such agent, atovaquone, collapses Ay in mitochondria from Plasmodium yoelii but has no effect on Ay of mammalian mitochondria (Srivastava et al., J. Biol. Chem. 272:3961-3966, 15 1997). Accordingly, the ET-based assay of Ay of the present invention can be used to screen libraries of compounds for novel antimalarial agents, i.e., compounds that cause Ay collapse in Plasmodium mitochondria but not in mammalian mitochondria. As another example, this embodiment of the invention is used to create and identify agents that selectively induce Ay collapse in mitochondria derived from 20 undesirable plants (e.g., weeds) but not in desirable plants (e.g., crops), or in undesirable insects (in particular, members of the family Lepidoptera and other crop damaging insects) but not in desirable insects (e.g., bees) or desirable plants. Such agents are expected to be useful for the management and control of such undesirable plants and insects. Cultured insect cells, including for example, the Sf9 and Sf21 cell 25 lines derived from Spodoptera frugiperda, and the HIGH FIVE T M cell line from Trichopolusia ni (these three cell lines are available from InVitrogen, Carlsbad, California) may be the source of mitochondria in certain such embodiments of the invention. In this embodiment of the invention, the subcellular compartment of 30 interest of a first species is loaded with a first donor-acceptor pair of molecules which WO 00/79274 PCT/USOO/17380 74 fluoresce at a first wavelength, and the corresponding subcellular compartment from a second species is loaded with a second donor-acceptor pair of molecules which fluoresce at a second wavelength. For example, mitochondria from two different species may be loaded with such donor-acceptor pairs of molecules. The two types of 5 loaded mitochondria are placed in a single chamber, and an agent to be tested for its ability to induce MPT in a species-specific manner is then also introduced into the chamber. The change in fluorescence at both the first and second wavelength is measured over time in a concomitant fashion. For example, a Fluorometric Imaging Plate Reader (FLIPRM) instrument (see infra) may be used to rapidly alternate between 10 a first mode, in which fluorescence at the first wavelength is monitored, to a second mode in which fluorescence at the second wavelength is monitored. A species-specific agent will induce MPT in the mitochondria from the first species, but not in those in the mitochondria from the second species, and will thus effect the degree, rate, frequency or extent in changes of fluorescence at one wavelength but not the other. 15 Diagnostics and Screening for Therapeutic Agents The invention may be used to develop assays of subcellular conditions or intracellular processes that are associated with diseases or disorders for a variety of purposes. One purpose is to aid in the diagnosis and prognosis of patients suffering from such diseases and disorders, and to help determine if an individual is potentially 20 predisposed to developing such diseases and disorders. Another purpose is to screen collections of compounds for agents having remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients suffering from, or potentially predisposed to developing, such diseases and disorders. The present invention therefore provides methods for identifying an 25 agent that alters cellular membrane potential, and that in certain preferred embodiments alters mitochondrial membrane potential. In certain other preferred embodiments the invention provides a method for identifying a regulator of an agent that alters mitochondrial membrane potential. The screening assays provided by the instant methods are thus directed in pertinent part to assaying, in the absence and presence of a WO 00/79274 PCT/USOO/17380 75 candidate agent or a candidate regulator, a cellular membrane potential by contacting a sample comprising one or more cellular membranes with an ET donor and an ET acceptor molecule, exciting the ET donor to produce an excited ET donor molecule, detecting a signal generated by energy transfer from the ET donor to the ET acceptor 5 and comparing the signal generated in the absence of the candidate agent (or regulator) to the signal generated in the presence of the candidate agent (or regulator). Embodiments that are directed to a method for identifying a regulator of an agent that alters mitochondrial membrane potential further comprise contacting a sample, prior to the step of detecting, with an agent that is either a known agent that alters mitochondrial 10 membrane potential or an agent that alters mitochondrial membrane potential and that is identified according to the methods provided herein. In those certain preferred embodiments wherein the invention is directed to a method for identifying an agent that alters mitochondrial membrane potential, or to a method for identifying a regulator of an agent that alters mitochondrial membrane 15 potential, neither the ET donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and the ET donor and the ET acceptor each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites as provided herein. Typically, based upon the teachings provided herein, a person having ordinary skill in the art can readily determine when a candidate agent alters a 20 cellular membrane potential such as mitochondrial membrane potential, for example, by detecting a statistically significant change in the membrane potential in the presence of the agent relative to the potential detected in the absence of the agent. Methods for determining mitochondrial membrane potential are also provided in U.S. application number 09/161,172. 25 Similarly, for purposes of determining whether a compound that is a candidate regulator of an agent that alters a cellular membrane potential such as mitochondrial membrane potential, methods for quantifying membrane potential will be useful. Agents that alter mitochondrial membrane potential include agents known to have such properties, including agents that dissipate mitochondrial membrane potential 30 and agents that collapse mitochondrial membrane potential (e.g., those described in WO 00/79274 PCT/USOO/17380 76 greater detail in the Examples below), as well as agents identified according to methods provided herein. A regulator of an agent that alters mitochondrial membrane potential includes any agent that in a specific manner directly or indirectly influences (e.g., increases or decreases) the ability of an agent that alters mitochondrial membrane 5 potential to alter mitochondrial membrane potential. Thus, for example, a regulator of an agent that alters mitochondrial membrane potential may be an agonist or may be an antagonist of the agent that alters mitochondrial membrane potential. For example, where an agent that alters mitochondrial membrane potential dissipates the potential, a regulator that is an agonist may potentiate such dissipation (e.g., cause collapse) while a 10 regulator that is an antagonist of the agent that alters mitochondrial membrane potential may confer a protective effect on mitochondrial membrane potential when the dissipating agent is present. Conversely, for an agent that alters mitochondrial membrane potential by preserving or enhancing AT,,, regulators that are agonists may also protect or enhance potential while regulators that are antagonists may lead to 15 dissipation or collapse of ATm. Without wishing to be bound by theory, a regulator as described herein may participate in intermolecular interaction events (e.g., recognition, binding, complex formation, covalent modification, alteration of conformation) with one or more of an agent that alters mitochondrial membrane potential and the subcellular target or targets of the agent that alters mitochondrial membrane potential, 20 including mitochondrial molecular components. (Mitochondrial molecular components are described, for example, in U.S. application number 09/161,172.) Thus, where a number of disorders and diseases result from processes involving mitochondria, the main energy source in cells of higher organisms, the invention provides compositions and methods for monitoring mitochondrial membrane 25 potential (Ay) and changes therein via energy transfer, as noted above. As described in detail herein, Ay is required for a variety of mitochondrial functions, and defects in the production or maintenance of Ay are associated with many diseases and disorders. Furthermore, changes in Ay occur in a variety of subcellular processes that can serve as targets for the development of therapeutic agents. Thus, the ET-based assay of Ay can 30 be used to help confirm the presence of a disease or disorder associated with alterations WO 00/79274 PCT/USOO/17380 77 in Ay in an individual, or an individual's predisposition to such a disease or disorder, and to screen for agents that stabilize, increase or decrease (as appropriate) Ay and can thus be used to treat such diseases and disorders. Moreover, the ET-based assay of AY can be used to screen for agents that selectively perturb Ay in undesirable cells such as, 5 e.g., cancer cells, thus leading to the specific destruction or inhibition of growth of such undesirable cells. Mitochondria provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes (for a review, see Ernster and Schatz, J Cell Biol. 91:227s-255s, 1981), including electron transport chain 10 (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis. In addition to their role in metabolic processes, mitochondria are also involved in the genetically programmed cell suicide sequence known as "apoptosis" (Green and Reed, Science 281:1309-1312, 15 1998; Susin et al., Biochim. et Biophys. Acta 1366:151-165, 1998). Defective mitochondrial activity, including but not limited to failure at any step of the elaborate multi-complex mitochondrial assembly, known as the electron transport chain (ETC), may result in (i) decreases in ATP production, (ii) increases in the generation of highly reactive free radicals (e.g., superoxide, peroxynitrite and 20 hydroxyl radicals, and hydrogen peroxide), (iii) disturbances in intracellular calcium homeostasis and (iv) the release of factors (such as such as cytochrome c and "apoptosis inducing factor") that initiate or stimulate the apoptosis cascade. Because of these biochemical changes, mitochondrial dysfunction has the potential to cause widespread damage to cells and tissues. 25 A number of diseases and disorders are thought to be caused by or be associated with alterations in mitochondrial metabolism and/or inappropriate induction or suppression of mitochondria-related functions leading to apoptosis. These include, by way of example and not limitation, chronic neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD); auto-immune diseases; 30 diabetes mellitus, including Type I and Type II; mitochondria associated diseases, WO 00/79274 PCTIUSOO/17380 78 including but not limited to congenital muscular dystrophy with mitochondrial structural abnormalities, fatal infantile myopathy with severe mtDNA depletion and benign "later-onset" myopathy with moderate reduction in mtDNA, MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke) and MIDD (mitochondrial 5 diabetes and deafness); MERFF (myoclonic epilepsy ragged red fiber syndrome); arthritis; NARP (Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE (Myopathy and external ophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy), LHON (Leber's Hereditary Optic Neuropathy), Kearns-Sayre disease; Pearson's Syndrome; PEO (Progressive External Ophthalmoplegia); Wolfram syndrome; DIDMOAD 10 (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, Deafness); Leigh's Syndrome; dystonia; schizophrenia; and hyperproliferative disorders, such as cancer, tumors and psoriasis. According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential 15 (AT) in the inner mitochondrial membrane by a coupled chemiosmotic mechanism. Conditions that dissipate or collapse this membrane potential, including but not limited to failure at any step of the ETC, may thus prevent ATP biosynthesis and hinder or halt the production of a vital biochemical energy source. Altered or defective mitochondrial activity may also result in a catastrophic mitochondrial collapse that has been termed 20 "mitochondrial permeability transition" (MPT). In addition, mitochondrial proteins such as cytochrome c and "apoptosis inducing factor" may dissociate or be released from mitochondria due to MPT (or the action of mitochondrial proteins such as Bax), and may induce proteases known as caspases and/or stimulate other events in apoptosis (Murphy, Drug Dev. Res. 46:18-25, 1999). 25 Defective mitochondrial activity may alternatively or additionally result in the generation of highly reactive free radicals that have the potential of damaging cells and tissues. These free radicals may include reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and potentially other reactive species that may be toxic to cells. For example, oxygen free radical induced lipid peroxidation 30 is a well established pathogenetic mechanism in central nervous system (CNS) injury WO 00/79274 PCTIUSOO/17380 79 such as that found in a number of degenerative diseases, and in ischemia (i.e., stroke). Mitochondrial involvement in the apoptotic cascade has been identified, for example mitochondrial release of cytochrome c, and may therefore be a factor in neuronal death that contributes to the pathogenesis of certain neurodegenerative (i.e., CNS) diseases. 5 There are, moreover, at least two deleterious consequences of exposure to reactive free radicals arising from mitochondrial dysfunction that adversely impact the mitochondria themselves. First, free radical mediated damage may inactivate one or more of the myriad proteins of the ETC. Second, free radical mediated damage may result in catastrophic mitochondrial collapse that has been termed "transition 10 permeability". According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential in the inner mitochondrial membrane by a coupled chemiosmotic mechanism. Free radical oxidative activity may dissipate this membrane potential, thereby preventing ATP biosynthesis and/or triggering mitochondrial events in the apoptotic cascade. Therefore, 15 by modulating these and other effects of free radical oxidation on mitochondrial structure and function, the present invention provides compositions and methods for protecting mitochondria that are not provided by the mere determination of free radical induced lipid peroxidation. For example, rapid mitochondrial permeability transition likely entails 20 changes in the inner mitochondrial transmembrane protein adenylate translocase that results in the formation of a "pore". Whether this pore is a distinct conduit or simply a widespread leakiness in the membrane is unresolved. In any event, because permeability transition is potentiated by free radical exposure, it may be more likely to occur in the mitochondria of cells from patients having mitochondria associated 25 diseases that are chronically exposed to such reactive free radicals. Altered mitochondrial function characteristic of the mitochondria associated diseases may also be related to loss of mitochondrial membrane electrochemical potential by mechanisms other than free radical oxidation, and such transition permeability may result from direct or indirect effects of mitochondrial genes, 30 gene products or related downstream mediator molecules and/or extramitochondrial WO 00/79274 PCTUSOO/17380 80 genes, gene products or related downstream mediators, or from other known or unknown causes. Loss of mitochondrial potential therefore may be a critical event in the progression of mitochondria associated or degenerative diseases. Diabetes 5 Diabetes mellitus is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing diabetes mellitus is reportedly maternally inherited, suggesting a mitochondrial genetic involvement (Alcolado et al., Br. Med. J. 302:1178-1180, 1991; Reny, Int. J. Epidem. 23:886-890, 1994). Diabetes is a heterogenous disorder with a strong genetic 10 component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals. At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis 15 and increased levels of reactive oxygen species. Studies have shown that diabetes mellitus may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. A small percentage of IGT individuals (5-10%) progress to insulin deficient 20 non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). These forms of diabetes mellitus, NIDDM and IDDM, are associated with decreased release of insulin by pancreatic beta cells and/or a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus 25 include obesity, vascular pathologies, peripheral and sensory neuropathies, blindness and deafness. Due to the strong genetic component of diabetes mellitus, the nuclear genome has been the main focus of the search for causative genetic mutations. However, despite intense effort, nuclear genes that segregate with diabetes mellitus are WO 00/79274 PCT/USOO/17380 81 known only for rare mutations in the insulin gene, the insulin receptor gene, the adenosine deaminase gene and the glucokinase gene. Accordingly, mitochondrial defects, which may include but need not be limited to defects related to the discrete non-nuclear mitochondrial genome that resides in mitochondrial DNA, may contribute 5 significantly to the pathogenesis of diabetes mellitus (Anderson, Drug Dev. Res. 46:67 79, 1999). A number of mitochondrial mutations associated with diabetic phenotypes have been described (for reviews, see Gerbitz et al., Biochim. Biophys. Acta 1271:253-260, 1995, or R6tig et al., Diabetes Metab. 22:291-298, 1996). A number of 10 such mutations occur in genes encoding factors involved in protein translation within mitochondria, such as mitochondrial tRNAs (see, e.g., Suzuki et al., Diabetes Care 17:1428-1432, 1994; Kishimoto et al., Diabetologia 38:193-200, 1995; van der Ouweland et al., Muscle Nerve Suppl. 3:S124-S130, 1995; Hanna et al., Am. J. Hum. Genet. 56:1026-1033, 1995; Sano et al., J. Neurol. 243:441-444, 1996; Kameoka et al., 15 Biochem. Biophys. Res. Commun. 245:523-527, 1998; and Hirai et al., J. Clin. Endocrinol. Metab. 83:992-994, 1998). Because mitochondrial translation is dependent on Ay (C6td et al., J. Biol. Chem. 264:8487-8490, 1989; Ctd et al., J. Biol. Chem. 265:7532-7538, 1990), alterations in Ay may result in diabetic phenotypes in some instances, and individuals suspected of having or being predisposed to developing 20 diabetes may be identified using the ET-based assay Ay of the invention. Furthermore, agents that increase and/or stabilize Ay are expected to have remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients suffering from, or thought to be predisposed to developing, diabetes. The ET based assay of Ay of the invention can also be used to estimate which agent(s) are most 25 likely to be effective for a given individual, in that a patient having mitochondria that exhibit an altered Ay is expected to be more likely to respond to agents that modulate Ay than a patient having mitochondria with a normal Ay.

WO 00/79274 PCT/USOO/17380 82 Parkinson's Disease Parkinson's disease (PD) is a progressive, chronic, mitochondria associated neurodegenerative disorder characterized by the loss and/or atrophy of dopamine-containing neurons in the pars compacta of the substantia nigra of the brain. 5 Like Alzheimer's Disease (AD), PD also afflicts the elderly. It is characterized by bradykinesia (slow movement), rigidity and a resting tremor. Although L-Dopa treatment reduces tremors in most patients for a while, ultimately the tremors become more and more uncontrollable, making it difficult or impossible for patients to even feed themselves or meet their own basic hygiene needs. 10 It has been shown that the neurotoxin 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) induces parkinsonism in animals and man at least in part through its effects on mitochondria. MPTP is converted to its active metabolite, MPP+, in dopamine neurons; it then becomes concentrated in the mitochondria. The MPP+ then selectively inhibits the mitochondrial enzyme NADH:ubiquinone oxidoreductase 15 ("Complex I"), leading to the increased production of free radicals, reduced production of adenosine triphosphate, and ultimately, the death of affected dopamine neurons. Apoptotic cell death is thought to constitute the terminal process in some neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. It has been proposed that agents that help to maintain Ay might offer novel agents for preventing or 20 treating neurodegenerative apoptosis (Tatton et al., Ann. Neurol. 44:S134-S141, 1998). Individuals suspected of having or being predisposed to developing Parkinson's disease (PD) may be identified using the ET-based assay Ay of the invention. Moreover, the ET-based Ay assay of the invention can be used to identify and characterize compounds that enhance or stabilize Ay, and these compounds are expected to have remedial, 25 therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients suffering from, or thought to be predisposed to developing, PD. The ET-based assay of Ay of the invention can also be used to estimate which agent(s) are most likely to be effective for a given individual, in that a PD patient having mitochondria that exhibit an altered Ay is expected to be more likely to respond to 30 agents that modulate Ay than a PD patient having mitochondria with a normal Ay.

WO 00/79274 PCT/USOO/17380 83 Alzheimer's Disease Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disorder that is characterized by loss and/or atrophy of neurons in discrete regions of the brain, and that is accompanied by extracellular deposits of p-amyloid and the 5 intracellular accumulation of neurofibrillary tangles. It is a uniquely human disease, affecting over 13 million people worldwide. It is also a uniquely tragic disease. Many individuals who have lived normal, productive lives are slowly stricken with AD as they grow older, and the disease gradually robs them of their memory and other mental faculties. Eventually, they cease to recognize family and loved ones, and they often 10 require continuous care until their eventual death. Mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1277 1287, 1995), and may be a cause of apoptotic cell death in neurons of the AD brain. Altered mitochondrial physiology may be among the earliest events in PCD (Zamzami 15 et al., J. Exp. Med 182:367-77, 1995; Zamzami et al., J. Exp. Med 181:1661-72, 1995) and elevated reactive oxygen species (ROS) levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994). Indeed, one hallmark pathology of AD is the death of selected neuronal populations in discrete regions of the brain. Cell death in AD is presumed to 20 be apoptotic because signs of programmed cell death (PCD) are seen and indicators of active gliosis and necrosis are not found (Smale et al., Exp. Neurolog. 133:225-230, 1995; Cotman et al., Molec. Neurobiol. 10:19-45, 1995.) The consequences of cell death in AD, neuronal and synaptic loss, are closely associated with the clinical diagnosis of AD and are highly correlated with the degree of dementia in AD (DeKosky 25 et al., Ann. Neurology 2757-464, 1990). In several cell types, including neurons, reduction in the mitochondrial membrane potential (AT) precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, mitochondrial, but not nuclear, enriched fractions are capable of inducing nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). 30 Moreover, cybrids comprising mitochondria derived from AD patients have lower WO 00/79274 PCT/USOO/17380 84 resting mitochondrial membrane potentials than the corresponding parental SH-SY5Y cell line, and cyclosporin A reverses the depressed Ay in the AD cybrids (Cassarino et al., Biochem. Biophys. Res. Commun. 248:168-173, 1998). Individuals suspected of having or being predisposed to developing AD may be identified using the ET-based 5 assay Ay of the invention. Moreover, the ET-based Ay assay of the invention can be used to identify and characterize compounds that enhance or stabilize Ay, and these compounds are expected to have remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients suffering from, or thought to predisposed to developing, AD. The ET-based assay of Ay of the invention 10 can also be used to estimate which agent(s) are most likely to be effective for a given individual, in that an AD patient having mitochondria that exhibit an altered AY is expected to be more likely to respond to agents that modulate Ay than an AD patient having mitochondria with a normal Ay. Other Neurological Disorders 15 Similar theories have been advanced for analogous relationships between mitochondrial defects and other neurological diseases, including Alzheimer's disease, Leber's hereditary optic neuropathy, schizophrenia, "mitochondrial encephalopathy, lactic acidosis, and stroke" (MELAS), and "myoclonic epilepsy ragged red fiber syndrome" (MERRF). 20 Increasing evidence points to the fundamental role of mitochondrial dysfunction in chronic neurodegenerative diseases (Beal, Biochim. Biophys. Acta 1366: 211-223, 1998), and recent studies implicate mitochondria for regulating the events that lead to necrotic and apoptotic cell death (Susin et al., Biochim. Biophys. Acta 1366: 151-168, 1998). Stressed (by, e.g., free radicals, high intracellular calcium, loss of 25 ATP, among others) mitochondria may release pre-formed soluble factors that can initiate apoptosis through an interaction with apoptosomes (Marchetti et al., Cancer Res. 56:2033-2038, 1996; Li et al., Cell 91:479-489, 1997). Release of preformed soluble factors by stressed mitochondria, like cytochrome c, may occur as a consequence of a number of events. In any event, it is thought that the magnitude of WO 00/79274 PCTUSOO/17380 85 stress (ROS, intracellular calcium levels, etc.) influences the changes in mitochondrial physiology that ultimately determine whether cell death occurs via a necrotic or apoptotic pathway. To the extent that apoptotic cell death is a prominent feature of degenerative diseases, mitochondrial dysfunction may be a critical factor in disease 5 progression. To the extent that Ay depression or collapse is a causative or compounding factor in degenerative disorders, individuals suspected of having or being predisposed to developing such disorders may be identified using the ET-based assay Ay of the invention. The ET-based Ay assay of the invention can also be used to identify and characterize agents that enhance or stabilize Ay, and these agents are 10 expected to have remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients suffering from, or thought to be predisposed to developing, such disorders. The ET-based assay of Ay of the invention can also be used to estimate which agent(s) are most likely to be effective for a given individual, in that a patient having mitochondria that exhibit an altered Ay is expected 15 to be more likely to respond to agents that modulate Ay than a patient having mitochondria with a normal Ay. Stroke In contrast to chronic neurodegenerative diseases, neuronal death following stroke occurs in an acute manner. A vast amount of literature now documents 20 the importance of mitochondrial function in neuronal death following ischemia/reperfusion injury that accompanies stroke, cardiac arrest and traumatic injury to the brain. Experimental support continues to accumulate for a central role of defective energy metabolism, alteration in mitochondrial function leading to increased oxygen radical production and impaired intracellular calcium homeostasis, and active 25 mitochondrial participation in the apoptotic cascade in the pathogenesis of acute neurodegeneration. A stroke occurs when a region of the brain loses perfusion and neurons die acutely or in a delayed manner as a result of this sudden ischemic event. Upon cessation of the blood supply to the brain, tissue ATP concentration drops to negligible WO 00/79274 PCTUSOO/17380 86 levels within minutes. At the core of the infarct, lack of mitochondrial ATP production causes loss of ionic homeostasis, leading to osmotic cell lysis and necrotic death. A number of secondary changes can also contribute to cell death following the drop in mitochondrial ATP. Cell death in acute neuronal injury radiates from the center of an 5 infarct where neurons die primarily by necrosis to the penumbra where neurons undergo apoptosis to the periphery where the tissue is still undamaged (Martin et al., Brain Res. Bull. 46:281-309, 1998). Much of the injury to neurons in the penumbra is caused by excitotoxicity induced by glutamate released during cell lysis at the infarct focus, 10 especially when exacerbated by bioenergetic failure of the mitochondria from oxygen deprivation (MacManus and Linnik, J. Cerebral Blood Flow Metab. 17:815-832, 1997). The initial trigger in excitotoxicity is the massive influx of Ca 2 primarily through the NMDA receptors, resulting in increased uptake of Ca 2 into the mitochondria (reviewed by Dykens, "Free radicals and mitochondrial dysfunction in excitotoxicity and 15 neurodegenerative diseases" in Cell Death and Diseases of the Nervous System, V. E. Koliatos and R.R. Ratan, eds., Humana Press, New Jersey, pages 45-68, 1999). The Ca2 overload collapses the mitochondrial membrane potential (ATm) and induces increased production of reactive oxygen species (Dykens, J Neurochem 63:584-591, 1994; Dykens, "Mitochondrial radical production and mechanisms of oxidative 20 excitotoxicity" in The Oxygen Paradox, K.J.A. Davies, and F. Ursini, eds., Cleup Press, U. of Padova, pages 453-467, 1995). If severe enough, AT. collapse and mitochondrial Ca 2 sequestration can induce opening of a pore in the inner mitochondrial membrane through a process called mitochondrial permeability transition (MPT), indirectly releasing cytochrome c and other proteins that initiate apoptosis (Bernardi et al., J Biol 25 Chem 267:2934-2939, 1994; Zoratti et al., Biochim Biophys Acta 1241:139-176, 1995; Ellerby et al., J Neurosci 17:6165-6178, 1997). Consistent with these observations, glutamate-induced excitotoxicity can be inhibited by preventing mitochondrial Ca 2 uptake or blocking MPT (Budd et al., J. Neurochem 66:403-411, 1996; White et al., J. Neurosci 16:5688-5697, 1996; Li et al., Brain Res 753:133-140, 1997; Stout et al., Nat. 30 Neurosci. 1:366-373, 1998).

WO 00/79274 PCT/USOO/17380 87 Agents and methods that maintain mitochondrial integrity during transient ischemia and the ensuing wave of excitotoxicity would be expected to be novel neuroprotective agents with utility in limiting stroke-related neuronal injury. Given the limited therapeutic window for blockade of necrotic death at the core of an 5 infarct, it may be particularly desirable to develop therapeutic strategies to limit neuronal death by preventing mitochondrial dysfunction in the non-necrotic regions of an infarct. As explained in more detail in Example 9 herein, such agents may be isolated by screening collections of compounds for their ability to stabilize Ay under excitotoxic conditions that mimic transient ischemia. Such agents are expected to have 10 remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease impeditive effects on patients who have had, or who are thought to be predisposed to have, strokes. The ET-based assay of Ay of the invention can also be used to estimate which agent(s) are most likely to be effective for a given individual, in that a patient having mitochondria that exhibit an altered AMJ is expected to be more likely to respond 15 to agents that modulate Ay than a patient having mitochondria with a normal Ay. Hyperproliferative Disorders Whereas mitochondria-mediated apoptosis may be critical in degenerative diseases, it is thought that disorders such as cancer involve the unregulated and undesirable growth (hyperproliferation) of cells that have somehow escaped a 20 mechanism that normally triggers apoptosis in such undesirable cells. Enhanced expression of the anti-apoptotic protein Bcl-2 and its homologues is involved in the pathogenesis of numerous human cancers. Bcl-2 acts by inhibiting programmed cell death and overexpression of Bcl-2, and the related protein Bcl-XL, block mitochondrial release of cytochrome c from mitochondria and the activation of caspase 3 (Yang et al, 25 Science 275:1129-1132, 1997; Kluck et al., Science 275:1132-1136, 1997; Kharbanda et al., Proc. Natl. Acad Sci. U.S.A. 94:6939-6942, 1997). In contrast, overexpression of Bcl-2 and Bcl-XL protect against the mitochondrial dysfunction preceding nuclear apoptosis that is induced by chemotherapeutic agents. In addition, acquired multi-drug resistance to cytotoxic drugs is associated with inhibition of cytochrome c release that is WO 00/79274 PCT/USOO/17380 88 dependent on overexpression of Bcl-XL (Kojima et al., J. Biol. Chem. 273: 16647 16650, 1998). There is a need for compounds and methods that inhibit the growth or enhance the death of cells and tissues that have escaped appropriate apoptotic signals, as 5 well as cytotoxic agents that cause the death of undesirable (e.g., cancer) cells by triggering the apoptotic cascade or otherwise. In particular, because mitochondria are mediators of apoptotic events, agents that stimulate mitochondrially mediated pro apoptotic events would be especially useful. Because mitochondria have been implicated in apoptosis, it is expected that agents that interact with mitochondrial 10 components will effect a cell's capacity to undergo apoptosis. Such agents are expected to have remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive effects on patients suffering from, or thought to be predisposed to developing, hyperproliferative diseases such as cancer and psoriasis. The ET-based assay of Ay of the invention can also be used to estimate which agent(s) are most likely 15 to be effective for a given individual, in that a patient having mitochondria that exhibit an altered Ay is expected to be more likely to respond to agents that modulate Ay than a patient having mitochondria with a normal Ay. The ET-based assay of mitochondrial Ay of the invention may also be used to identify agents that are selectively cytotoxic for hyperproliferative or other 20 undesirable cell types. For example, Ay is elevated in some carcinoma cell lines, and agents that accumulate in mitochondria as a function of Ay (such as rhodamine 123) are preferentially cytotoxic to such carcinoma cells (Modica-Napolitano et al., Cancer Res. 47:4361-4365, 1987; Andrews et al., Cancer Res. 52:1895-1901, 1992). In sum, the invention may be used to develop assays for subcellular 25 conditions or intracellular processes, such as changes in mitochondrial Ay, in order to identify and characterize agents to treat degenerative disorders and diseases as well as hyperproliferative diseases. The ET-based assay of Ay can be used to identify, depending on the disease or disorder for which treatment is sought, agents that are mitochondria protecting agents, anti-apoptotic agents or pro-apoptotic agents.

WO 00/79274 PCT/USOO/17380 89 The following examples illustrate the invention and are not intended to limit the same. Those skilled in the art will recognize, or be able to ascertain through routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of 5 the present invention.

WO 00/79274 PCT/USOO/17380 90 EXAMPLES EXAMPLE I SELECTION OF COMPOUNDS AND OPTIMIZATION OF CONDITIONS FOR ET-BASED ASSAYS 5 In order to develop an ET-based assay to detect conditions within a subcellular compartment (such as an organelle or a membrane-bounded portion thereof), and monitor changes thereof, it is necessary to determine appropriate pairs of donor and acceptor compounds and useful concentrations thereof. Using the information and methods presented herein, one skilled in the art can readily determine donor and 10 acceptor -compounds, and concentrations thereof, appropriate for a variety of such assays. One step in the process of developing an ET-based assay involves optimizing concentrations of the donor and acceptor compounds, as well as other conditions for the assay. In general, with regards to the concentrations of the donor and 15 acceptor compounds, at least two criteria apply. First, the concentrations of the donor and acceptor compounds should be sufficient for energy transfer to occur. Second, the concentration of each compound should be low enough that (a) any non ET-based signal from the compounds is negligible, so that the background signal in the assay is minimal, and (b) any undesirable effects on cellular physiology, including cellular 20 toxicity, and/or effects on the subcellular compartment of interest, are minimal. It should be noted, however, that not every compound will have undesirable effects on cellular physiology. In the case of a FRET-based assay of Ay using NAO and TMRM, these criteria are applied as follows. Because NAO is known to be toxic to certain cells at 25 higher concentrations, for example at > 10 pM as reported by Maftah et al. (FEBS Lett. 260:236-240, 1990), NAO sensitivity of cells to be used should first be determined to avoid exposing cells to toxic levels of this ET molecule. The NAO concentration that is toxic may vary depending on the cell (e.g., a cell line) selected for use in a given experiment, and on other conditions such as duration of exposure to NAO, the presence WO 00/79274 PCTIUSO0/17380 91 of other toxic or protective factors are present, and the like. The person having ordinary skill in the art will, however, be able to select and readily determine suitable NAO concentrations and/or durations of NAO exposure to cells without undue experimentation, based on the present disclosure. 5 For instance, a series of experiments was performed to determine the optimum ratio and concentrations of NAO and TMRM for a FRET-based assay of Ay. These tests can be applied to other pairs of donor and acceptor compounds in other ET based assays. General Protocols 10 The FRET-based assay of Ay was generally carried out in the following manner, although variations to these general procedures can be made without affecting the sensitivity, accuracy or efficiency of the assay. Cell Lines and Preparation Thereof A variety of cell lines were used in the following experiments. The 15 neuroblastoma SH-SY5Y is a multiply subcloned cell line of human origin (Perez-Polo et al., Dev. Neurosci. 5:418-423, 1982). SH-SY5Y is a well-characterized cell line that is capable of differentiating into neuron-like cells, and is an accepted cellular model for a variety of neuronal cell functions (for reviews, see, e.g., Vaughan et al., Gen. Pharmacol. 26:1191-1201, 1995; Pahlman et al., Acta Physiol. Scand Suppl. 592:25 20 37, 1990). Cybrid (cytoplasmic hybrid) cells comprise a nuclear component from one cell type and a cytoplasmic (including mitochondrial) component from another cell type. Procedures for preparing cybrid cells, derived from mitochondrial DNA (mtDNA) depleted (rho' or p 0 ) cells, and comprising mitochondria derived from patients having 25 Alzheimer's disease, have been previously described (Miller et al., J. Neurochem. 67:1897-1907, 1996; Swerdlow et al., Neurology 49:918-925, 1997; and U.S. Patent No. 5,888,498, all of which are hereby incorporated by reference). The 1685 cybrid cell line is one example of a cybrid cell line of this type. The 1685 cybrid cell line was WO 00/79274 PCT/USOO/17380 92 created by fusing platelets from an AD donor with SH-SY5Y neuroblastoma cells that had been made rho' by extended treatment with ethidium bromide. "MixCon" designates a Mixed Control composed of cybrids prepared in like fashion but using platelets from n normal age-matched patients (n = 2-3, depending on the particular 5 experiment) for the construction of the cybrid cells. NCI-H460 is a human lung large cell carcinoma cell line available from the American Type Culture Collection (ATCC, Manassas, VA) under accession No. ATCC HTB-177. A preferred cellular medium for NCI-H460 cells is 90% (RPMI 160 medium with 2 mM L-glutamine, 1.5 g/L sodium carbonate, 4.5 g/L glucose, 10 mM 10 HEPES and 1.0 mM sodium pyruvate), 10 % (fetal bovine calf serum). MCF-7 is a human breast carcinoma cell line available from the ATCC under accession No. ATCC HTB-22. MCF-7 has been used in studies of the relationship between disruption of mitochondrial Ay and apoptotic events (see, e.g., Heerdt et al., Cancer Res. 59:1584-1591, 1999). A preferred cellular medium for MCF 15 7 cells is 90% MEM (minimum essential Eagle's medium supplemented with 2 mM L glutamine and Earle's BSS, 1.5 g/L sodium carbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate), 10 % FBS-ins (fetal bovine calf serum with 10 ptg/ml bovine insulin). In general, cells were plated at about 2 to 3 x 104 cells per well on 96 20 well microplates (CostarTM, black wall; clear, flat bottom) at least about 24 hours prior to the assay. HBSS was generally used as cellul medium, but any media appropriate for a given cell line may be used in the assay. Preparation of Donor and Acceptor Compounds A 5 mg/ml stock solution of the ET donor compound, nonylacridine 25 orange (NAO, Molecular Probes, Inc., Eugene, OR; catalog #A1372), was prepared in DMSO. The stock solution was aliquoted into microfuge tubes and stored frozen at -20'C until thawed on ice immediately prior to the assay. Unless otherwise specified, for use in assays the 5 mg/ml NAO stock solution was diluted 1:5000 in Hank's WO 00/79274 PCT/USOO/17380 93 balanced salt solution (HBSS, Life Technologies, Grand Island, NY) to yield a working stock solution containing 1 tg/ml NAO, which was further diluted as indicated below. A stock solution of acceptor compound, 100 mM TMRM (Molecular Probes, Inc., Eugene, OR; catalog #T668), was prepared in DMSO. This concentration 5 corresponds to 20,000 X the final concentration used in the assay. The stock solution was aliquoted into microfuge tubes and stored frozen at -20'C until thawed on ice immediately prior to the assay. A combined stock solution was also prepared for ease of manipulation, containing both the ET donor and acceptor compounds (25 mM TMRM and 1 mg/ml 10 NAO) in DMSO (i.e., both ET molecules at 5,000 times the final concentration used in the assay). The combined stock solution was aliquoted into microfuge tubes and stored frozen at -20*C, and thawed on ice immediately prior to the assay. Instrument Preparation The FLIPRTM heaters and laser were turned on for at least 1 hour before 15 the assay is performed. Typically, the following settings were used: shutter, 0.4 sec.; f stop, 0.2; filter, #2; laser at 300 mW (15 A). In later experiments, a special order filter (Omega Optical, Inc., Brattleboro, VT) for 530 + 25 nm was used. In the FLIPRTM instrument, there are positions for three 96-well microplates. A centrally located 96-well microplate contains samples, and up to two 20 96-well plates, one on each side of the central plate, containing additional reagents can be included. In a typical assay, the first reagent 96-well (8 rows, 12 columns) plate was set up so that the wells in Row A contained media (typically, HBSS), the wells in Row B contained a Ay collapsing agent (typically, CCCP), and the remaining Rows (C through H) contained the test compound(s) (e.g., candidate agents). Furthermore, in a 25 Type II assay (see Example 5), a second reagent plate was set up so that each well contained an appropriate amount of a Ay collapsing agent (typically, CCCP) to be added to the samples sometime after the test compound(s).

WO 00/79274 PCT/USOO/17380 94 Fluorophore Loading Fifteen minutes prior to the assay, the entire plate was gently flicked over a sink to remove the cell media. The displaced media was replaced in each well with 100 pl HBSS that contained 5 pM TMRM and was prewarmed to 37*C. In 5 general, it was preferred to prewarm media and reagents to 37'C and to maintain cells at 37'C in order to avoid thermal shock that can itself cause changes in Ay or cause the death of sensitive cells. Ten minutes later, 20 pil of the NAO working stock solution prepared as described above (1 ptg/ml NAO) was added to each well (final concentration, 200 ng/ml, equal to 0.4 tM). 10 In an optional step, after letting the cells incubate in the presence of both fluorophores for about 5 minutes, excess fluorophore was removed by gently flicking the plate to remove cell media and adding 100 ul prewarmed HBSS to each well; this process was repeated up to three times. After the final plate flicking, 100 pl prewarmed HBSS was added to each well. Depending on cell type used in a particular experiment, 15 the cells could be incubated for varying periods of time prior to addition of the test compound(s) (e.g., candidate agents), with no appreciable loss of ET signal. In the case of SH-SY5Y cells, this incubation period was up to approximately 40 minutes. Assay Readings Prior to the addition of test compounds, about 20 readings were taken on 20 the FLIPR instrument at 3-second intervals. Although these data were not used in calculating the results of the assay, they were useful for assessing the integrity of the cells and/or monitoring for spontaneous collapse of Ay. For example, if cellular integrity was compromised, a significant collapse in Ay would be detected after the optional rinsing step but before addition of the test compounds. Next, the test 25 compounds were added and 175 readings were taken at 5-second intervals. To determine the ET value corresponding to maximal collapse of Ay (i.e., Ay ~ 0 in theory), a Ay collapsing agent (e.g., CCCP) was added as follows. In Type I assays (Figure 3A), the collapsing agent was added to wells distinct from those receiving the test compounds at roughly the same time that the test compounds were WO 00/79274 PCTIUSOO/17380 95 added, and readings of these wells were taken at the same time as readings were taken of the wells that received test compounds. In Type II assays (Figure 3B), the collapsing agent was added to the same wells that received the test compounds after readings had been taken at 5-second intervals for a period of time (typically about 9-15 minutes), and 5 readings were then taken for a second period of time roughly equivalent to the first period of time. Data Analysis The assay results are presented as plots of relative fluorescence units (RFU) over time (Figure 6) for qualitative analysis. For quantitative analyses, 10 calculations were as follows: For Type I assays, the initial instrument reading for each well was set to zero. The readings taken at 5-second intervals following those taken at 3-second intervals to verify cellular integrity, typically readings numbered from about reading 21 to about reading 195-200, were summed (EF,). Tests of significance for multiple (i.e., 15 >2) groups, such as one-way ANOVA of treatment groups with no transform, Newman Keuls or Bonferroni (Dunn's) multi-comparisons, were used to evaluate the significance of results. For Type II assays, the initial instrument reading for each well was set to zero, and readings taken at 5-second intervals (following integrity confirmation as 20 described above) numbered from about 21 to about 195-200 were summed (IF). For normalization, the readings during the final 4 minutes (i.e., readings numbers about 214 to 230) after addition of the Ay collapsing agent (CCCP) to maximally compromise membrane potential were averaged (FCCCp). Because the use of ratios would violate mathematical assumptions inherent in ANOVA algorithms, the data were transformed 25 (log or arcsin) before being evaluated for significance in one-way ANOVA analyses. For either type of assay, sums and averages for each well were calculated using the software provided with the FLIPRTM instrument, exported into EXCELTM (Microsoft, Inc., Redmond, WA) via *.txt, and finally exported into GB Stat (Dynamic Microsystems, Silver Springs, MD) for ANOVA. FLIPRTM kinetic data are exported WO 00/79274 PCT/USOO/17380 96 into EXCEL for mean and standard error calculations of the readings taken over the time courses. It was desirable (but not necessary) to back up all FLIPRTM data on CD, or another appropriate machine-readable format, on a daily basis. Results 5 In an initial set of experiments, MixCon cybrid cells were treated with six different concentrations of TMRM (0, 1.25, 2.5, 5.0, 10 and 20 p.M) and NAO (0, 6, 12, 25, 50, 100, 200 and 400 ng/ml; respectively, 0, 13, 26, 52, 105, 210, 420 and 840 nM) on a 96-well plate. Each of the 48 possible combinations of TMRM and NAO concentrations was tested in duplicate using a FLIPR instrument using a 0.1 second 10 shutter with the laser set at 300 mW and readings taken using a 510-590 nm filter. CCCP was added to all samples (1.5 tM) at 1 minute after the plate was put into the FLIPRTM instrument. According to non-limiting theory, if FRET occurred between NAO and TMRM, which localize to the inner mitochondrial membrane and the mitochondrial 15 matrix, respectively, then a change in FRET-based signal should occur following CCCP addition. Thus, the addition of CCCP would cause Ay to be decreased and, as a consequence, the mitochondrial concentration of the acceptor compound (TMRM) would also decrease as TMRM exited the mitochondria and/or was taken up to a lesser degree by mitochondria. Because the donor compound (NAO) is retained by 20 mitochondria regardless of Ay, the donor and acceptor compounds would cease to be in sufficient proximity to one another for FRET to occur, and the signal resulting from FRET should decrease, as indicated by a change in fluorescence (expressed in relative fluorescence units, RFU). Also according to non-limiting theory, the energy transfer from NAO to 25 TMRM can be measured either directly or indirectly (see Figure 1). Direct measurement of NAO -> TMRM FRET involves (a) exciting the donor, NAO, at an appropriate wavelength for its excitation [XD(ex)], which in turn emits energy at a wavelength [XD(em)] that overlaps the excitation spectrum of the acceptor, TMRM, and (b) measuring the emission from excited TMRM molecules at or near their peak WO 00/79274 PCT/USOO/17380 97 emission wavelength [XA(em)]. Indirect measurement of NAO -> TMRM FRET also involves exciting NAO at XDex, but it is the emission from the donor NAO, not from the acceptor TMRM, that is measured (i.e., XD(em) is measured rather than XA(em)). If energy transfer occurs efficiently from the excited donor (NAO) to the acceptor 5 (TMRM), then emissions from the donor will be "quenched" and the signal at XD(em) will be minimal. If and when the acceptor compound ceases to be proximal to the donor, energy transfer will cease to occur and the emissions from the donor will be "dequenched" (i.e., the signal at XD(em) will increase). In the present example, FRET was measured indirectly. TMRM + NAO 10 loaded SY5Y cells were exposed to light of wavelength 488 nm (near XD(ex) for NAO, 485 nm) and the signal at 530 + 25 nm (near XD(em) for NAO) was measured over time after CCCP addition. The prediction is that, if FRET occurs between the donor NAO and the acceptor TMRM, the addition of CCCP (which results in a decreased concentration of TMRM in the mitochondria) should yield a dequenching of the signal 15 from NAO (i.e., increasing fluorescence at or near XDem). In contrast, if FRET had been measured directly, the signal at or near XD(em) for TMRM would have been measured, and would be expected to decrease following the addition of CCCP and a resultant TMRM exodus from mitochondria. The results of indirect FRET measurement are shown in Figure 4. In 20 these results, FRET was seen as an increase in signal (dequenching of NAO emission) that occurred following CCCP addition only when both donor and acceptor compounds were present at a given set of concentrations, i.e., the increase did not occur when either the acceptor or donor compound alone was present at the same concentration. For example, in Figure 4, FRET occurred in wells E9, E10, F9, FlO, 9G and 10G, as 25 contrasted with the signals in wells A9 and AlO (NAO absent) and wells F1 and F2 (TMRM absent). Although FRET was probably occurring in other wells in the extreme upper right-hand portion of Figure 4, the signal in these wells may also include a significant background signal component derived from NAO alone (e.g., compare wells H 11 and H12 to HI and H2) or TMRM alone (e.g., compare wells H 11 and H12 to All 30 and A12). Based on these results, useful preferred concentrations of NAO and TMRM WO 00/79274 PCTUSOO/17380 98 for the assay include 50 ng/ml NAO and 10 ptM TMRM (wells E9 and E10), 100 ng/ml NAO and 10 pM TMRM (wells F9 and F1O), 200 ng/ml NAO and 10 pLM TMRM (wells G9 and G10), and 200 ng/ml NAO and 5 pM TMR (wells G7 and G8). The data in Figure 4 were analyzed as described above for the Type I 5 assay, i.e., the initial reading for each well was set to zero, and the RFU readings taken from about 21 to about 175 seconds were summed (EF). The results for varying concentrations of TMRM were graphed as a function of NAO concentration, as shown in Figure 5. FRET occurred at 50, 100 and 200 ng/ml of NAO with 5 or 10 pM TMRM, as evidenced by the increase in signal at these concentrations (e.g., compare the 10 5 and 10 tM TMRM curves in the 50-200 ng/ml NAO range with the 0, 1.25 and 2.5 pM TMRM curves in the same range of NAO concentrations). In another experiment designed to examine the background signal from each fluorophore individually as well as time course of CCCP-mediated Ay collapse, FRET was measured in cells treated with either 5 p.M TMRM , 420 nM NAO, or with 15 both compounds, for a more extended period after CCCP addition (1.5 p.M). As shown in Figure 6, a rapid increase in fluorescence occurred within the first two minutes after CCCP addition, after which the change in fluorescence reached a plateau. When either NAO or TMRM was present alone, the fluorescent signal was essentially constant. In order to determine if the NAO dequenching signal that was measured 20 in the FRET-based assay might be linear over different cell densities, the following experiments were performed. Different numbers of MixCon or 1685 cybrid cells were preincubated in replicate in wells of 96-well plates for about 10 minutes with 5 pM TMRM, after which 4 ng/ml NAO was added and the cells were incubated for an additional 5 minutes. Finally, CCCP (1 pM) was added to each well, and fluorescence 25 signals were monitored at 530 + 25 nm using a FLIPRTM device. Mitochondrial efflux of TMRM then took place, as evidenced by an increase in fluorescence signal corresponding to the dequenching of NAO emissions over time. The initial slopes of the curves (RFU over time) were plotted against the number of cells per well. The results showed that the Ay-dependent fluorescent signal increased in a linear fashion 30 over the range of from about 38,000 to about 330,000 cells per well.

WO 00/79274 PCT/USOO/17380 99 Although many of the experiments described herein made use of a FLIPR instrument, and involved a series of measurements over time, the invention may be carried out using any instrument or device of sufficient sensitivity and capable of monitoring at least two time points (i.e., before and after addition of an agent that 5 affects Ay). In one experiment, for example, MixCon and 1685 cybrid cells were preincubated with TMRM and NAO as above, and fluorescence at 538 nm was measured using anfmaxTM (Molecular Devices, Inc., Sunnyvale, CA) fluorimetric plate reader (excitation = 485 nm) and then treated with CCCP (final concentration, 1.3 ptM). Ten minutes later, the fluorescence at 538 nm was again determined, and found to have 10 increased significantly as compared to control cells treated with buffer alone, in all three cell types (SY5Y cells, MixCon cybrids, 1685 cybrids). Moreover, the 1685 cybrid cell line, which comprises mitochondria from a patient having Alzheimer's disease, was more sensitive to ionomycin, i.e., showed a greater degree of loss of Ay than the control cybrid cells (MixCon) or the parental SH 15 SY5Y cell line. This result demonstrates that the assay can be used to detect differences among cell types in reactions to agents that influence Ay. EXAMPLE 2 PARAMETER-DEPENDENT Co-LOCALIZATION OF ACCEPTOR-DONOR COMPOUNDS 20 Another step in the process of developing an ET-based assay to detect conditions within a subcellular compartment (such as an organelle or a membrane bounded portion thereof), and monitor changes thereof, is to confirm that not only do the donor and acceptor compounds co-localize in sufficient proximity for energy transfer to occur, but also that such co-localization is dependent on the state of the 25 parameter to be measured. That is, at least one of the compounds must localize to (accumulate in) the subcellular compartment of interest as a function of the measured parameter, and must leave that compartment and/or accumulate less rapidly or efficiently in that compartment as that parameter changes. For example, for an ET-based assay designed to measure Ay of 30 mitochondria, one of the compounds (either the donor or the acceptor) must accumulate WO 00/79274 PCT/USOO/17380 100 in and/or be released from mitochondria in a manner that is dependent on Ay, whereas the presence of the other compound (the acceptor or donor, respectively) in mitochondria must be Ay-independent. Combining these criteria with the information presented herein, one skilled in the art can readily choose donor-acceptor combinations 5 that are appropriate for ET-based Ay assays. Compounds whose mitochondrial concentration is not dependent on Ay include, by way of example and not limitation, NAO (Petit et al., Eur. J. Biochem. 194:389-397, 1990; Maftah et al., Biochem. Biophys. Res. Comm. 164:185-190, 1989), MitoTracker@ Green FM (U.S. Patent Nos. 5,459,268 and 5,686,261), MitoFluor T M 10 Green (Haugland, Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., Spence, ed., Molecular Probes, inc., Eugene, Oregon, 1996, page 269) and fusion proteins comprising (a) a red- or yellow-shifted Green Fluorescent Protein polypeptide, or a "FLASH" polypeptide, and (b) a polypeptide sequence that localizes the fusion protein to the mitochondrial matrix or inner membrane. These compounds are listed as 15 Group IV and V donor compounds in Table 2. A series of representative Group IV and V acceptor compounds is also presented in Table 2. Of the Group IV and V acceptor compounds in Table 2, those that accumulate in mitochondria in a Ay-dependent manner include, by way of example and not limitation, rhodamine 123 (Emaus et al., Biochim. Biophys. Acta 850:436-448, 1986; Scaduto et al., Biophys. J. 76:469-477, 20 1999), TMRM and TMRE (Farkas et al., Biophys. J 56:1053-1069, 1989; Ehrenberg et al., Biophys. J 53:785-794, 1988). With regard to specific sites of accumulation of these compounds, NAO specifically interacts with the inner mitochondrial membrane (Maftah et al., Biochem. Biophys. Res. Comm. 164:185-190, 1989). Without wishing to be bound by theory, 25 TMRM, TMRE and rhodamine 123 are believed to localize to the mitochondrial matrix, although a recent report indicates that these compounds additionally accumulate reversibly in the inner and outer aspects of the inner mitochondrial membrane, possibly as a function of localized microenvironments there featuring intensified membrane potential (Scaduto et al., Biophys. J 76:469-477, 1999). Accordingly, irrespective of 30 whether TMRM, TMRE and rhodamine 123 localize to the inner mitochondrial WO 00/79274 PCTUSOO/17380 101 membrane or the mitochondrial matrix (or both), they are expected to be in close proximity to the inner mitochondrial membrane, where NAO localizes (see Figure 1). In order to confirm that FRET only occurs between appropriately localized donor-acceptor pairs of compounds in living cells, the following experiment 5 was carried out. SH-SY5Y cells were cultured and assayed as in Example 1 with the following exceptions. Cells were incubated with an "acceptor" compound at 5 IM for 10 minutes, and then further incubated with a "donor" compound at 4 ng/ml for an additional 10 minutes. At this time, an agent that collapses AY, CCCP, was added to the cells at a concentration of 1 ptM, and relative fluorescence was measured using an 10 fmaxTM (Molecular Devices, Inc., Sunnyvale, CA) fluorimetric plate reader (excitation, 485 nm; emission read at 538 nm + 20 nm). The mean rate of change in relative fluorescent units (RFU) in 6 to 8 replicate wells was calculated as the slope of the curve over the initial 3.5 minutes using the software provided with thefmaxTM instrument via least squares linear regression. 15 The results are shown in Table 4. FRET was detected between NAO and TMRM, which localize to the inner mitochondrial membrane and the mitochondrial matrix, as indicated by the mean rate of RFU change following CCCP addition. FRET occurred between NAO and TMRM until the addition of CCCP, which caused a decrease in Ay and exit of the acceptor compound (TMRM) from mitochondria. 20 Because the donor compound (NAO) is retained by mitochondria independent of AY, the donor and acceptor compounds ceased to be in sufficient proximity to one another for FRET to occur, and the signal resulting from FRET declined (as indicated by the relatively rapid rate of change in RFU). In contrast to the effect seen with NAO and TMRM, when calcein or 25 CO-Fluor were used as "donor" compounds, the rate of RFU change following CCCP addition was negligible. This reflects the fact that, although calcein and CO-Fluor have emission peaks similar to that of NAO, they did not localize to mitochondria and thus were not in close enough proximity to the "acceptor" compound (the mitochondrially localized TMRM) for FRET to occur. In like fashion, when SNAFL, which does not 30 localize to mitochondria, was used as an "acceptor" compound and NAO was used as a WO 00/79274 PCTIUS00/17380 102 "donor" compound, FRET was not observed, even though the excitation peak wavelength of SNAFL (514 nm) is closer to emission peak wavelength of NAO (517 nm) than the excitation peak wavelength of TMRM (544 nm). Thus, as expected, for energy transfer to occur, both spectral overlap and physical proximity were required. 5 Table 4: FRET Only Occurs Between Appropriately Co-Localized Donor and Acceptor Compounds "Donor" Compound "Acceptor" Compound Mean Rate kD(ex) kD(em) XA(ex) XA(em) of RFU Change NAO 495 nm 519 nm 548 nm 573 nm TMRM 0.3750*** Calcein 494 nm 517 nm 548 nm 573 nm TMRM 0.0050*** CO-Fluor 492 nm 517 nm 548 nm 573 nm TMRM 0.0025*** NAO 495 nm 519 nm 514 nm 546 nm SNAFL 0.0025*** In sum, energy transfer (in this example, FRET) occurred only when the 10 ET donor and acceptor molecules were appropriately co-localized within the subcellular compartment of interest. Moreover, processes that caused an ET donor or ET acceptor molecule to localize to a different site in such a manner that the pair of ET molecules were no longer in sufficient proximity for energy transfer to occur were monitored and assayed by measuring changes in a signal generated as a result of the energy transfer. 15 EXAMPLE 3 PARAMETER-DEPENDENT CHANGES IN ENERGY TRANSFER The preceding Examples show how to determine energy transfer between an ET donor and an ET acceptor molecule, how to optimize ET assay conditions including concentrations of the donor and acceptor compound, and how to demonstrate 20 that energy transfer is dependent upon co-localization of both compounds within the same or adjacent subcellular sites. In order to demonstrate that an ET-based assay detects the condition or parameter within a subcellular compartment of interest, and WO 00/79274 PCTUSOO/17380 103 monitor changes thereof, it is useful to validate the assay with agents having known effects on the chosen condition or parameter. Using a FRET-based assay designed to measure Ay of mitochondria as a model, a variety of agents are known in the art to lower (dissipate) or eliminate 5 (collapse) Ay. Additionally, some agents are known to increase Ay above normal levels, i.e., to hyperpolarize mitochondria. Both types of agents were evaluated using the FRET-based assay of Ay. Agents that Increase AI Oligomycin is an example of a compound that hyperpolarizes 10 mitochondria. MixCon cybrid cells were contacted with TMRM (5 tM) and NAO (420 nM) as in Example 1. On the same 96-well plate, a second set of MixCon cells was also treated with 10 pM oligomycin, dissolved in HBSS buffer for 10 minutes prior to addition to cells, and added to cells 10 minutes before the addition of TMRM. The "initial FRET signal," i.e., the first reading before initiating Ay collapse, was 15 determined for eight separate wells of each of the three combinations of cells and agents using a FLIPRTM instrument. If the agents work as expected, and according to non-limiting theory, hyperpolarization should increase Ay, leading to increased intramitochondrial TMRM accumulation, leading in turn to increased energy transfer (i.e., NAO quenching). The 20 results (Table 5) show that oligomycin had the predicted effect. That is, because the cells treated with oligomycin contained hyperpolarized mitochondria, the initial FRET signal was significantly less than that seen in cells that were not exposed to oligomycin. Table 5: Effect of Oligomycin on FRET-Based Assay of Ay Cells Oligomycin Initial FRET Significance* Relative to Standard Signal MixCon, No Oligomycin Error MixCon (none) 521.1 - 17 MixCon 10 uM 296.2 P < 10-8 13 WO 00/79274 PCT/USOO/17380 104 * Calculated via two-tailed t-test Agents that Decrease Av and Protective Agents: lonomycin and Bongkrekic Acid The effect on Ay of the calcium ionophore ionomycin, which dissipates 5 and eventually collapses Ay, alone or in combination with bongkrekic acid (BKA), was compared to the effects of the Ay-collapsing agent CCCP. Because BKA binds to the adenine nucleotide translocator, the activity of which is required for mitochondrial permeability transition (MPT), it was predicted that BKA would have an ameriolating effect on the Ay dissipation caused by ionomycin. SH-SY5Y cells were treated with 10 donor and acceptor compounds (respectively, NAO, 420 nm, and TMRM, 5 ptM) according to the procedure described in Example 1, and HBBS media, CCCP (1.5 piM), ionomycin (5 iM), or ionomycin (5 ptM) and BKA (2 iM; preincubated with cells at 37'C for 10 minutes before TMRM was added). RFU was monitored using a FLIPR T M instrument. 15 The results (Figure 7) show that, as in the preceding Examples, CCCP (Fig. 7, "C") induced a rapid increase in fluorescence, apparently due to dequenching of the NAO emission signal and consistent with collapse of Ay and exodus from the mitochondria of the acceptor compound, TMRM. Treatment with ionomycin (Fig. 7, "I") ultimately yielded a more gradual change in fluorescence, as was expected for an 20 agent known in the art to cause a slower dissipation in Ay than CCCP. The addition of BKA to ionomycin-treated cells (Fig. 7, "I+BKA") moderated the effect of ionomycin effects and ultimately resulted in a fluorescence signal that was similar to that seen when HBSS media only (Fig. 7, "MO") was added to the cells. lonomycin and Ruthenium Red 25 Ruthenium red was confirmed to have a protective effect with regard to the Ay-dissipating effects of ionomycin. Ionomycin is an ionophore that increases the level of cytosolic calcium; this leads to a dissipation of Ay as mitochondria take up WO 00/79274 PCTUSOO/17380 105 calcium from the cytosol. Ruthenium red blocks the activity of the mitochondrial calcium uniporter, thus inhibiting or blocking mitochondrial uptake of calcium. Ruthenium red would therefore be expected to counteract the effect of ionomycin. SH SY5Y cells were prepared and preincubated with NAO and TMRM as in the preceding 5 examples and treated with CCCP (1.5 pM), ionomycin (5 pM) with ruthenium red (100 iM) and media (HBSS) only. Fluorescence was measured over time at 530 + 25 nm using a FLIPRTM instrument. The results (Figure 8) demonstrate that the FRET-based assay yielded data that follow the expected patterns, i.e., the ionomycin-mediated dissipation of Ay was essentially completely blocked by ruthenium red. 10 lonomycin or MPP+ and Cyclosporin A In another related experiment, cyclosporin A was confirmed to have a protective effect with regard to the Ay-dissipating effects of ionomycin. Cyclosporin A binds to cyclophilin D and, like BKA, blocks MPT, and is thus expected to counteract the effect of ionomycin. MixCon cells were prepared and preincubated with NAO and 15 TMRM as in the preceding examples, and treated with ionomycin (5 [pM). One group of cells was preincubated with cyclosporin A (10 pM) for 15 minutes prior to CCCP addition. Fluorescence was measured over time at 530 + 25 nm using a FLIPRTM instrument. The results (Figure 9) demonstrate that the FRET-based assay yields data that follow the expected patterns, i.e., the ionomycin-mediated dissipation of Ay was 20 inhibited by cyclosporin A. In other experiments, the assay was used to confirm that cyclosporin A (10 pLM, added 10 minutes prior to addition of the Ay agent) essentially blocked the long-term (> 10 minutes after addition) dissipation and collapse of Ay otherwise caused by 0.5 mM MPP+. Atractyloside and Cyclosporin A 25 The FRET assay described above and in the preceding examples was also validated by the fact that it showed a dissipation of Ay in SH-SY5Y cells treated with atractyloside (ATR, 5 mM) that peaked at about 6 minutes after ATR addition. At this concentration of ATR, Ay recovered after about 15 minutes, whereas CCCP (1 WO 00/79274 PCT/USOO/17380 106 pM) led to a more complete collapse of Ay that was maintained for at least 15 minutes. Pretreatment with cyclosporin A (5 ptM, 5 minutes) resulted in a significant moderation of the response to ATR; the peak fluorescent signal in the ATR-plus-cyclosporin A sample was roughly half that of the sample treated with ATR alone. 5 In sum, energy transfer (in this example, FRET) occurred in a manner that accurately reflected changes in a parameter (in these examples, Ay) known to influence the concentration of the donor and/or acceptor compounds (in this example, the concentration of the acceptor compound TMRM decreased as a function of decreasing Ay). Moreover, the measured activities of agents known to increase (e.g., 10 oligomycin) or decrease (e.g., CCCP. ionomycin, MPP+, ATR) the chosen parameter (Ay) were in agreement with their predicted effects. The same was true for protective agents (BKA, ruthenium red, cyclosporin A) that are known to counteract, in whole or in part, the effects of parameter-changing agents. These results indicate that the ET based assay may be used to screen for and evaluate previously uncharacterized 15 compounds for their effects on the chosen parameter (in this example, AY) and for their ability to counteract the effects of known compounds on the parameter of interest. EXAMPLE 4 EVALUATION OF ASSAY RESULTS The results presented in the preceding examples demonstrate the need to 20 evaluate ET-based assay results in a fashion that yields meaningful conclusions. Using the results presented in Figure 7 as an example, although the initial rates of change in RFU of the samples treated with CCCP, ionomycin or ionomycin and BKA were similar from about 78 seconds to about 127 seconds, the readings for these three samples diverged thereafter and were markedly different at 460 seconds. There are a 25 variety of ways to evaluate the results of an ET-based assay, as summarized in Table 6, for example, using the results shown in Figure 7. One method for evaluating ET-based assays is to measure the time taken in each sample to reach a defined RFU value, i.e., to determine a threshold intercept value for each sample. Such a determination will indirectly reflect the initial slope of 30 the curves. As shown in Table 6, however, selection of an appropriate threshold WO 00/79274 PCT/USOO/17380 107 intercept RFU value is critical in this method of evaluation. Selecting RFU=2220, for example, as the intercept yields results that are inconsistent with the expected effects on Ay of the various treatments (i.e., CCCP > ionomycin > ionomycin & BKA > media only). Moreover, the RFU=2220 results are also somewhat confounding as the sample 5 treated with ionomycin and BKA intercepts RFU=2220 twice. On the other hand, selecting a lower intercept value (RFU=160) yields results having the expected order. In the latter case, however, the protective effects of BKA might not be fully appreciated, as the result for ionomycin plus BKA (0.345) is only slightly different than that for ionomycin alone (0.400). 10 Another method of evaluation of ET-based assays is to directly determine the initial slope of the curve for each sample. However, as the results shown in Figure 7 demonstrate, data from different samples can yield curves having similar initial slopes, even thought the overall shapes of the curves and their endpoints are distinct. 15 Another method of evaluation is to sum the area under the curve of the plot, or to undertake some similar operation such as, e.g., adding the RFU values of each time point, for each sample over a given time frame. As shown in Table 6, this method yields results for the four treatments that are consistent with the expected order of effect on Ay (i.e., CCCP > ionomycin > ionomycin & BKA > media only). Thus, 20 summing the area under each curve, or performing an operation that yields results that correspond to the area under the curves, is preferable in most instances, although other methods of evaluation may be used. Table 6: Different Evaluations of the Results in Figure 7 Treatment Area Time 2220 Time 160 (from Ratio to Time Ratio to Time Ratio to expected Area media (min.) to media (min.) to media most to least Under only Reach RFU only Reach RFU only effect on Curve * Sample = 2220 ** Sample = 160 ** Sample Ay) I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ WO 00/79274 PCT/USOO/17380 108 CCCP 16.2 x 105 10.1 0.85 0.133 0.050 0.083 ionomycin 6.79 x 10 5 4.22 0.55 0.086 0.200 0.333 ionomycin 1 = 0.75 1 = 0.117 and BKA 2.37 x 10 5 1.47 2 = 1.85 2 = 0.711 0.255 0.425 media only 1.61 x 10 5 1.00 6.40 1.000 0.600 1.000 * Measured as sum of all readings over 0 to 460 seconds. ** Measured from moment when all 4 curves were coincident (t= 1.1 min.). EXAMPLE 5 FRET-BASED ASSAY OF Ay 5 The preceding Example illustrates a potential limitation in the "Type I" FRET-based assay of Ay, in which the effects of various agents on Ay were compared to the effects of an agent (CCCP) that collapses Ay (Figure 3A). In order to yield more meaningful results, the "Type II" assay was developed. In the Type II assay, the agent(s) being tested is first added to a sample and, after allowing the agent(s) being 10 tested some time to exert their effects, a Ay collapsing agent is subsequently added to the same sample in order to drive Ay to zero, thus establishing a baseline value for the results. Figure 3B shows a Type II assay. In one version of the Type II assay, wherein a compound is being tested for its ability to dissipate Ay, the symbols in Figure 15 3B are as follows. Optional initial readings ("A" or "B") that can be normalized to zero are first taken. The candidate Ay-dissipating compound is added at timepoint "2." If the candidate Ay-dissipating compound has little or no effect on Ay, a signal like that represented by the solid line ("C"') is expected, whereas a test compound that dissipates Ay results in a signal like that represented by the dashed line ("C"). At timepoint "3," 20 an agent that completely collapses Ay (e.g., CCCP) is added, and a reading ("D") is taken after the collapse ofAy is complete, in order to allow for normalization of the various samples for variations in cell density, and in efficiency of loading of the ET WO 00/79274 PCT/USOO/17380 109 donor and acceptor compounds. The Ay-dissipating activity of the test compound is calculated as the Ay-Dissipating Value according to the formula: Ay-Dissipating Value = (C-B) / (D-B) 5 wherein a higher value for the Ay-Dissipating Value indicates a greater Ay-dissipating ability of the candidate compound. In another version of the Type II assay, wherein a compound is being tested for its ability to inhibit or enhance the activity of an agent that dissipates AY, the 10 symbols in Figure 3B are as follows. An optional initial reading ("A") that can be normalized to zero is first taken. The test compound is added at timepoint "1 ," and a baseline measurement ("B") is taken. The Ay-dissipating agent (e.g., ionomycin, atractyloside, etc.) is added at timepoint "2." If the test compound has little or no effect on the activity of the Ay-dissipating agent, a signal like that represented by the dotted 15 line ("C") is expected, whereas a test compound that inhibits or protects against the activity of the Ay-dissipating agent results in a signal like that represented by the solid line ("C' "). At timepoint "3," an agent that completely collapses Ay (e.g., CCCP) is added, and a reading ("D") is taken after the collapse ofAy is complete in order to allow for normalization for variations in cell density and efficiency of loading of the donor 20 and acceptor compounds. The activity of the test compound is calculated as the Efficacy Index according to the formula: Efficacy Index = (C-B) / (D-B) 25 wherein a lower value for the Efficacy Index indicates a greater protective effect of the test compound. Although CCCP and ionomycin are used in the following exemplary experiments, other Ay collapsing agents are known and can be used. Such Ay collapsing agents include, by way of example and not limitation, valinomycin, A23187 30 and 4-Br-A23187. It is desirable to establish a dose-response curve for whatever Ay collapsing agent is used, as conditions for the Type II assay are preferably such that Ay WO 00/79274 PCT/USOO/17380 110 collapses, and the measured signal reaches a plateau, in a rapid manner (i.e., preferably within 5 minutes after addition of the Ay collapsing agent, more preferably within 3 minutes, and most preferably within 2 minutes). Another parameter that can be established from dose-response experiments is the optimal concentration of AY 5 collapsing agent. A dose-response curve for CCCP is shown in Figure 10. In the experiments performed to generate the data in this figure, SH-SY5Y cells were treated with 420 nM NAO and 5 pM TMRM according to the general procedure of Example 1 and then monitored for approximately 60 seconds before the indicated amount of CCCP 10 was added. Dequenching of the emission signal from NAO was measured as in the preceding Examples. The dose-response curve reveals an increasingly rapid loss of NAO dequenching, as evidenced by the increasingly rapid rise in RFU, as higher concentrations of CCCP are used. These data also suggest that 10 pLM was a near saturating concentration of CCCP to use, as the response to 10 pM CCCP was only 15 slightly greater than that seen when 5 pM CCCP was applied (compare to the change in responses between 2.5 uM and 5 uM CCCP). The results from a representative Type II FRET experiment are shown in Figure 11, which shows relative fluorescence units + standard errors for readings taken at the indicated timepoints. In this experiment, SH-SY5Y cells were contacted with 20 NAO and TMRM according to the procedure of Example 1, and placed in a FLIPR instrument. After about 2 minutes, half the samples were treated with prewarmed media alone and the other half were treated with prewarmed media comprising 5 piM of the Ay-dissipating agent 4-bromo-A23187. About 6.5 minutes later, the Ay-collapsing agent CCCP (final concentration, 5 pM) was added to all the samples and the 25 fluorescence was read for an additional 7.5 minutes. As shown in Figure 11, the cells treated with 4-bromo-A23187 ("4-BR") exhibited a gradual loss of AY up until the time CCCP was added, at which point Ay further decreased and ultimately collapsed. As also shown in Fig. 11, the cells treated with media ("MO") also showed a rapid loss of Ay following CCCP addition and approached complete Ay collapse, the MO and 4-BR WO 00/79274 PCT/USOO/17380 111 curves becoming asymptotic after about 600 seconds and for the remainder of the experiment. EXAMPLE 6 DOSE RESPONSE CURVES FOR Ay-DSSIPATING AND AY PROTECTIVE AGENTS 5 Having established the basic parameters of the ET-based assay of Ay, more exact experiments were carried out to demonstrate that the assay can be used to generate dose-response curves for both Ay-dissipating and Ay-protective agents. SH SY5Y cells were used in these experiments. The calcium ionophore ionomycin was used as a mock candidate agent compound being evaluated for its capacity to cause 10 dissipation of Ay, and cyclosporin A was used as a mock ionomycin-protective agent. Cells were grown to specific cell density and transferred to 96-well plates as described above. For both sets of experiments, TMRM and NAO were added at the concentrations and in the order and timing described in Example 1. For the experiments involving ionomycin alone, ionomycin was added at various 15 concentrations 10 minutes after addition of NAO. In the case of the experiments designed to quantify the ability of cyclosporin A to protect against the effects of ionomycin, cells were loaded for 10 minutes with TMRM and for 5 minutes with NAO as described above for fluorophore (ET donor and acceptor molecules) loading, following which the cells were washed and exposed to various concentrations of 20 cyclosporin A for 15 minutes prior to initiation of instrument readings. Readings numbered 1-21 were recorded at 3-second intervals, and thereafter readings numbered 22-196 were recorded at 5-second intervals. As shown in Figure 13, the sum of the fluorescence signal over each time interval was determined and plotted against the log(1) ionomycin concentration (M) to generate a cyclosporin A dose-response curve. 25 The dose response curves for cells exposed to ionomycin in three separate experiments (50,000 cells per well in each experiment) are shown in Figure 12. The data generated parallel curves when plotted, demonstrating the reproducibility of the assay in analyzing compounds have a negative impact on Ay. The dose response curves for cells pretreated with varying amounts of 30 cyclosporin A and then exposed to ionomycin in three separate experiments (39,000 WO 00/79274 PCT/USOO/17380 112 cells per well in each experiment) are shown in Figure 13. The data generated similar curves when plotted, demonstrating the reproducibility of the assay in analyzing compounds that protect mitochondria from agents that have a negative impact on Ay. EXAMPLE 7 5 FRET IN VARIOUS CELL TYPES In the preceding examples, the FRET-based assay of Ay was performed on a neuroblastoma cell line (SH-SY5Y), and on the MixCon and 1685 cybrid cell lines that are generated from p 0 SY5Y cells. Although the control (MixCon) and Alzheimer's (1685) cybrids show the same general response to various agents and 10 treatments that influence Ay, some differences were detected by the FRET-based assay. In this example, MixCon or 1685 cells (about 50,000 cells per well) were preincubated with 420 nM NAO and 5 [tM TMRM according to the procedure of Example 1, after which the calcium ionophore A23187 (0 to 5 tM) was added. Detectable loss of quenching of the NAO signal (i.e., fluorescence at 530 + 25 nm) was measured over 15 time (4 minutes). The results are expressed as sums of all the datapoints over the 4 minute windows for each concentration of A23187 (Figure 14) and reveal some differences between the SH-SY5Y parental cells and the 1685 and MixCon cybrids. The AD (1685) cybrids demonstrated the highest degree of sensitivity to A23187, and the 20 control (MixCon) cybrids were somewhat more sensitive to A23187 than the parental SH-SY5Y cells. Statistical analysis (ANOVA) demonstrates that the increased susceptibility of the AD (1685) cybrid cells was significant. Thus, the ET-based assay of Ay of the present invention can be used to characterize mitochondrial abnormalities in whole cells. When such cells are isolated from an individual suspected of having or 25 being predisposed to having a mitochondria-associated disease (e.g., a disease associated with altered mitochondrial function), the assay may be used to aid in the diagnosis of such diseases.

WO 00/79274 PCT/USOO/17380 113 EXAMPLE 8 ET-BASED ASSAYS FOR DETECTING SPECIFIC CELL TYPES IN A SAMPLE Assays utilizing energy transfer can be used to detect specific cell types in a biological sample. For example, rhodamine 123 (a Group II, III and IV acceptor 5 compound; see Table 2) is taken up rapidly and retained for long periods (greater than 24 hours) by a variety of human carcinoma cells after washing, even though it is not usually well retained by other cell types when they are washed (Nadakavukaren et al., Cancer Res. 45:6093-6099, 1985; Summerhayes et al., Proc. Natl. Acad. U.S.A. 79:5292-5296, 1982; Christman et al., Gynecol. Oncol. 39:72-79, 1990). 10 An ET-based assay for carcinoma cells in a sample thus comprises the steps of (1) obtaining a biological sample from a patient, wherein the sample comprises cells (e.g., including carcinoma cells); (2) contacting the cells in the sample with rhodamine 123; (3) optionally washing the cells; (4) contacting the cells with a mitochondrial donor compound from Group II, III or IV (Tables 2 and 3), such as NAO, 15 MitoTracker@ Green FM or MitoFluor T M Green; (5) exciting the sample with light having a wavelength within the excitation spectrum of the donor, and (6) detecting energy transfer as a quenching of the donor emission by rhodamine-123. Carcinoma cells retain rhodamine 123 and thus exhibit FRET with the donor compound. The following experiment was carried out in order to demonstrate that 20 certain cell types (in this Example, a human carcinoma cell line) differentially take up and retain particular ET donor and/or acceptor molecules as provided herein, and therefore have unique properties permitting such specific cell types to be detected by an ET-based assay of the present invention, thereby distinguishing such cell types from others that may be present. NCI-H460 is a human lung large cell carcinoma cell line 25 (see Example 1 for details). NCI-H460 cells were added to 96-well plates (about 50,000 cells per well). In a Type II Ay assay TMRM (5 ptM) and NAO (420 nM) were added to the cells according to the procedure of Example 1. lonomycin (50 piM) in media was also added to one set (n = 24) of samples and media only was added to a control set of samples. The Ay collapsing agent CCCP (5 ptM) was added to all the WO 00/79274 PCT/USOO/17380 114 samples about 9 minutes later. Fluorescence was measured using a FLIPRTM instrument during the experiment, as described above. The results are shown in Figure 15. Although ionomycin ("I") caused a large degree of Ay dissipation, the carcinoma cells recovered relatively rapidly by about 5 6 minutes after addition of ionomycin. This recovery was unlike that seen with the cybrid cell lines or the neuroblastoma SH-SY5Y cell line used in the preceding Examples, and suggested that the mitochondria in the carcinoma cell line take up TMRM more rapidly, either in general or at least after a challenge to Ay, than did mitochondria from other cell types. It is particularly noteworthy that differential 10 susceptibility to inducers of AT collapse, as shown here by differential sensitivity to ionomycin detected in the FRET assay of mitochondrial membrane potential, can be used to distinguish cell types: The ionomycin concentration used here for NCI-H460 cells (50 ptM), a concentration from which these cells recovered, was ten times the ionomycin concentration to which SH-SY5Y cells were sensitive, as indicated by their 15 loss of mitochondrial membrane potential (Fig. 12). As described above, Figure 12 depicts increased dequenching of NAO fluorescence at higher ionomycin conditions using SH-SY5Y cells, indicative of greater mitochondrial membrane potential collapse at the higher ionomycin concentrations, which effected the loss of TMRM from the mitochondrial compartment. 20 EXAMPLE 9 METHODS FOR IDENTIFYING COMPOUNDS FOR TREATING STROKE Mechanisms of cell death from ischemia and reperfusion involve both necrosis and delayed apoptosis, with mitochondrial dysfunction as a common antecedent to both. A number of events follow ischemia-induced loss of mitochondrial 25 function, including decreased mitochondrial energy metabolism, increased mitochondrial production of toxic reactive oxygen species (ROS) after reperfusion, and active mitochondrial initiation of apoptotic cascades in conditions where energy production can be restored. Following a neuronal ischemic event, mitochondrial ATP production 30 halts due to the lack of oxygen. Although glycolytic ATP production can continue WO 00/79274 PCT/USOO/17380 115 under anoxic conditions, glycolysis cannot meet the energy demands of neurons due to limited stores of glycolysis substrates in the brain. Still, lactate does accumulate in anoxic brain tissue, providing a measurable endpoint for biologic assays. Because of losses in aerobic competence, the tissue ATP concentration drops to negligible levels 5 within minutes after cessation of oxygen flow to the brain. Without adequate ATP, the ATP-dependent ion transporters fail, and the loss of ion homeostasis results in osmotic lysis and necrosis of neurons at the anoxic core of the infarct. De-energization also involves the loss of ATP-dependent transport processes that sequester glutamate. Massive influx of Ca' and other ions ensues from 10 activation of voltage-dependent and ligand-dependent ion channels (White et al., J. Neurosci. 15:1318-1328, 1995; Harrington et al., Neuron 16:219-228, 1996; Schinder et al., J Neurosci. 16:6125-6133, 1996). Upon reperfusion, high levels of cytosolic Ca 2 directly activate mitochondrial calcium uptake, preventing the establishment of normal mitochondrial function upon re-introduction of oxygen. Excessive Ca accumulation 15 in the mitochondria can potentiate the production of oxygen-and carbon-centered radicals in neurons and lead to inactivation of mitochondrial electron transfer system (Dykens, J. Neurochem. 63:584-591, 1994; Reynolds et al, J. Neurosci. 15:3318-3327, 1995; Dugan et al., J. Neurosci. 15:6377-6388, 1995, Bindokas et al., J. Neurosci. 16:1324-1336, 1996). 20 Another consequence of mitochondrial Ca uptake is the induction of the membrane permeability transition (MPT), the opening of a nonspecific, voltage sensitive, pore that dissipates ATm and allows solutes of <1,500 Daltons to equilibrate across the inner mitochondrial membrane (see reviews, Zoratti et al., Biochim. Biophys. Acta 1241:139-176, 1995; Bernardi et al., J. Bioenerg. Biomemb. 26:509-517, 1994). 25 High ATm that is normally generated by the electron transport chain in the absence of high Ca' or free radical-induced injury, is a potent deterrent to MPT pore formation. Agents that moderate MPT and ATm collapse, such as Bcl-2 and cyclosporin A, correspondingly moderate glutamate excitotoxicity both in vitro and in vivo (Hoyt et al., Br. J Pharmacol. 122:803-808, 1997; Niemninen et al., Neurosci. 75:993-997, 1996; WO 00/79274 PCT/USOO/17380 116 Ankarcrona et al., FEBS Lett. 394:321-324, 1996; Uchino et al., Acta Physiol. Scand 155:469-471, 1995; Li et al., Brain Res. 753:133-140, 1997). Failure of cellular Ca 2 ' efflux mechanisms and activation of phospholipases and proteases appear as late-stage events after ischemia and can lead to 5 widespread damage to membranes and proteins. Cells exposed to less severe stress may initiate an apoptotic cascade. In this case, mitochondria may be reversibly damaged and release sufficient levels of apoptogenic factors to induce death while maintaining a residual capacity to generate ATP (MacManus et al., J. Cerebral Blood Flow Metab. 17:815-832, 1997). Therefore, healthy mitochondria play a bifunctional role in 10 preservation of neuronal viability in ischemia/reperfusion injury: 1) by supplying ATP, mitochondria provide the driving force for glutamate re-uptake from the synaptic cleft and the ATP-dependent maintenance of normal membrane potential that further resists opening of voltage-sensitive ion channels, and 2) uninjured mitochondria resist the release of factors that can direct neurons down an apoptotic pathway. Maintaining 15 mitochondrial integrity during ischemia/reperfusion and thereby defending against the ensuing wave of excitotoxicity thus permits identification of novel neuroprotective agents having utility for preventing stroke-related neuronal injury. Primary Screening Assays Measurement of ATm provides a comprehensive indication of 20 mitochondrial function and integrity. Therefore, the primary screening assay in stroke drug discovery utilizes the ET-based assay of AT in whole cells in a high-throughput format. Agents and methods that maintain mitochondrial integrity during transient ischemia and the ensuing wave of excitotoxicity are expected to be effective neuroprotective agents with utility in limiting stroke-related neuronal injury. Given the 25 limited therapeutic window for blockade of necrotic death at the core of an infarct, it is particularly desirable to develop therapeutic strategies to limit neuronal death by preventing mitochondrial dysfunction in the non-necrotic regions of an infarct. To this end, compounds are screened for their effects on AT under control and Ca 2 overload conditions.

WO 00/79274 PCT/USOO/17380 117 Following a stroke, much of the injury to neurons in the penumbra is caused by excitotoxicity induced by glutamate released during cell lysis at the infarct focus. In order to more closely mimic in vivo biochemical and cellular events, primary screening assays are carried out in cells comprising one or more types of glutamate 5 receptors (for reviews, see Gasis et al., Curr. Opin. Neurobiol. 1:20-26, 1991; Westbrook, Curr. Opin. Neurobiol. 4:337-346, 1994; and Lynch et al., Curr. Opin. Neurobiol. 7:510-516, 1994). Glutamate receptors include ionotropic glutamate receptors (iGluRs) and metabotropic receptors (mGluRs). The iGluRs are glutamate-gated cation channels that 10 are further classified further into the subclasses of NMDA receptors, AMPA receptors and kainate receptors. NMDA receptors are heteromeric complexes including, for example, NMDAR1/2A, NMDARI/2B, NMDAR1/2C and NMDARI/2D. AMPA receptors are homomeric complexes including, for example, GluR1, GluR2, GluR3 and GluR4. Kainate receptors may be either homomeric or heteromeric complexes of 15 GluR5, GluR6, GluR7, KA-1 and KA-2. The mGluRs are 7-transmembrane G-protein coupled receptors that are also classified further into subclasses. Some mGluRs are phospholipase C-coupled mGluRs that increase cytosolic calcium; these include mGluRl and mGluR5. Other mGluRs are adenylate cyclase-coupled mGluRs that decrease cytosolic cAMP; these include mGluR2, mGluR3, mGluR4, mGluR7, and 20 mGluR8. One example of a cell comprising one or more types of glutamate receptors that are used in primary screens is a primary cortical neuron expressing endogenous NMDA receptors. In these cells, application of extracellular glutamate elevates intracellular calcium levels (Stout et al., Nat. Neurosci. 1:366-373, 1998). 25 Subsequent to glutamate addition, changes in AT are measured using the ET-based assay of AT. Mitochondria-defective cybrid cells that have a depressed AT (Cassarino et al., Biochem. Biophys. Res. Commun. 248:168-173, 1998) are also utilized in addition to primary neuronal cultures in order to provide a more extensive response to agents and/or conditions that are tested for their ability to dissipate or collapse AT.

WO 00/79274 PCT/USOO/17380 118 Other examples of cells comprising one or more types of glutamate receptors that are used in primary screens include cells that have been genetically engineered to express or overexpress one or more glutamate receptors. A number of mammalian cell lines have been manipulated to stably express glutamate receptors in 5 culture (for a review, see Varney et al., Methods. Mol. Biol. 128:43-59, 1999). Non limiting examples of glutamate receptors that have been cloned and expressed in mammalian cells include NMDRA1A/2A and NMDARIA/2B (Varney et al., J. Pharmacol. Exp. Ther. 279:367-378, 1996); NMDAR2C, isoforms 1, 2, 3 and 4 (Dagget et al., J. Neurochem. 71:1953-1968, 1998); GluR3 (Varney et al., J 10 Pharmacol. Exp. Ther. 285:358-370, 1998); and GluRlb and GluR5a (Lin et al., Neuropharmacology 36:917-931, 1997). Secondary Screening Assays Compounds that prevent the prolonged collapse of ATm caused by high [Ca 2 1i] in the primary assay are evaluated further in secondary assays, including ROS 15 production, measurement of cytochrome c release and caspase-3 activation as indicators of apoptosis, and cell viability. In this way, "hits" identified in the FRET ATm assay are further verified, and the mechanism by which the compound affects ATm can be better defined. The rationale for these assays is based on evidence suggesting that compounds that can maintain mitochondrial integrity under conditions of excitotoxicity 20 or oxidative stress may correspondingly decrease the release of apoptogens and rescue penumbral neurons that are at risk of apoptotic death following transient ischemia. The following assays are described in more detail in copending U.S. patent application Serial No. 09/299,044, filed April 23, 1999. Assay for Inhibition of Production of Reactive Oxygen Species Using 25 Dichlorofluorescin Diacetate: According to this assay, the ability of a mitochondria protecting agent of the invention to inhibit production of ROS intracellularly may be compared to its antioxidant activity in a cell-free environment. Production of ROS may be monitored using, for example by way of illustration and not limitation, 2',7' dichlorodihydroflurescein diacetate ("dichlorofluorescin diacetate" or DCFC), a WO 00/79274 PCTIUSOO/17380 119 sensitive indicator of the presence of oxidizing species. Non-fluorescent DCFC is converted upon oxidation to a fluorophore that can be quantified fluorimetrically. Cell membranes are also permeable to DCFC, but the charged acetate groups of DCFC are removed by intracellular esterase activity, rendering the indicator less able to diffuse 5 back out of the cell. In the cell-based aspect of the DCFC assay for inhibition of production of ROS, cultured cells may be pre-loaded with a suitable amount of DCFC and then contacted with a mitochondria protecting agent. After an appropriate interval, free radical production in the cultured cells may be induced by contacting them with iron 10 (III)/ ascorbate and the relative mean DCFC fluorescence can be monitored as a function of time. In the cell-free aspect of the DCFC assay for inhibition of production of ROS, a mitochondria protecting agent may be tested for its ability to directly inhibit iron/ ascorbate induced oxidation of DCFC when the protecting agent, the fluorescent 15 indicator and the free radical former are all present in solution in the absence of cells. Comparison of the properties of a mitochondria protecting agent in the cell-based and the cell-free aspects of the DCFC assay may permit determination of whether inhibition of ROS production by a mitochondria protecting agent proceeds stoichiometrically or catalytically. Without wishing to be bound by theory, 20 mitochondria protecting agents that scavenge free radicals stoichiometrically (e.g., on a one-to-one molecular basis) may not represent preferred agents because high intracellular concentrations of such agents might be required for them to be effective in vivo. On the other hand, mitochondria protecting agents that act catalytically may moderate production of oxygen radicals at their source, or may block ROS production 25 without the agents themselves being altered, or may alter the reactivity of ROS by an unknown mechanism. Such mitochondria protecting agents may "recycle" so that they can inhibit ROS at substoichiometric concentrations. Determination of this type of catalytic inhibition of ROS production by a mitochondria protecting agent in cells may indicate interaction of the agent with one or more cellular components that synergize 30 with the agent to reduce or prevent ROS generation. A mitochondria protecting agent WO 00/79274 PCT/USO0/17380 120 having such catalytic inhibitory characteristics may be a preferred agent for use according to the method of the invention Mitochondria protecting agents that are useful according to the instant invention may inhibit ROS production as quantified by this fluorescence assay or by 5 other assays based on similar principles. The person having ordinary skill in the art is familiar with variations and modifications that may be made to the assay as described here without departing from the essence of this method for determining the effectiveness of a mitochondria protecting agent, and such variations and modifications are within the scope of this disclosure. 10 Assay for Mitochondrial Permeability Transition (MPT) Using 2-,4 Dimethylaminostyryl-N-Methylpyridinium (DASPMI): According to this assay, one may determine the ability of a mitochondria protecting agent of the invention to inhibit the loss of mitochondrial membrane potential that accompanies mitochondrial dysfunction. As noted above, maintenance of a mitochondrial membrane potential may 15 be compromised as a consequence of mitochondrial dysfunction. This loss of membrane potential or mitochondrial permeability transition (MPT) can be quantitatively measured using the mitochondria-selective fluorescent probe 2-,4 dimethylaminostyryl-N-methylpyridinium (DASPMI). Upon introduction into cell cultures, DASPMI accumulates in 20 mitochondria in a manner that is dependent on, and proportional to, mitochondrial membrane potential. If mitochondrial function is disrupted in such a manner as to compromise membrane potential, the fluorescent indicator compound leaks out of the membrane bounded organelle with a concomitant loss of detectable fluorescence. Fluorimetric measurement of the rate of decay of mitochondria associated DASPMI 25 fluorescence provides a quantitative measure of loss of membrane potential, or MPT. Because mitochondrial dysfunction may be the result of reactive free radicals such as ROS, mitochondria protecting agents that retard the rate of loss of DASPMI fluorescence may be effective agents for treating mitochondria associated diseases according to the methods of the instant invention.

WO 00/79274 PCT/USOO/17380 121 Assays of Apoptosis in Cells Treated with Mitochondria Protecting Agents: As noted above, mitochondrial dysfunction may be an induction signal for cellular apoptosis. According to the assays in this section, one may determine the ability of a mitochondria protecting agent of the invention to inhibit or delay the onset 5 of apoptosis. Mitochondrial dysfunction may be present in cells known or suspected of being derived from a subject with a mitochondria associated disease, or mitochondrial dysfunction may be induced in cultured normal or diseases cells by one or more of a variety of physical (e.g., UV radiation), physiological and biochemical stimuli with which those having skill in the art will be familiar. 10 Apoptosis and/or biochemical processes associated with apoptosis may also be using one or more "apoptogens," i.e., agents that induce apoptosis and/or associated processes when contacted with or withdrawn from cells or isolated mitochondria. Such apoptogens include by way of illustration and not limitation (1) apoptogens that are added to cells having specific receptors therefor, e.g., tumor 15 necrosis factor (TNF), FasL, glutamate and NMDA; (2) withdrawal of growth factors from cells having specific receptors for such factors, such factors including, for example, IL-3 or corticosterone; and apoptogens that may be added to cells but which do not require a specific receptor, including (3) Herbimycin A (Mancini et al., J. Cell. Biol. 138:449-469, 1997), (4) Paraquat (Costantini et al., Toxicology 99:1-2, 1995); (5) 20 ethylene glycols (http://www.ulaval.ca/vrr/rech/Proj/532866.html); (6) protein kinase inhibitors, such as, e.g.: Staurosporine, Calphostin C, d-erythro-sphingosine derivatives, Chelerythrine chloride, Genistein, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, KN 93, Quercitin, N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide and caffeic acid phenethyl ester; (7) ionophores such as, e.g.: lonomycin and valinomycin; 25 (8) MAP kinase inducers such as, e.g.: Anisomycin and Anandamine; (9) cell cycle blockers such as, e.g.: Aphidicolin, Colcemid, 5-fluorouracil and homoharringtonine; (10) Acetylcholinesterase inhibitors such as, e.g.: berberine; (11) anti-estrogens such as, e.g.: Tamoxifen; (12) pro-oxidants, such as, e.g., tert-butyl peroxide and hydrogen peroxide; (13) free radicals such as, e.g., nitric oxide; (14) inorganic metal ions, such as, 30 e.g.: cadmium; (15) DNA synthesis inhibitors such as, for example, Actinomycin D, WO 00/79274 PCT/USOO/17380 122 Bleomycin sulfate, Hydroxyurea, Methotrexate, Mitomycin C, Camptothecin, daunorubicin and intercalators such as, e.g., doxorubicin; (16) protein synthesis inhibitors such as, e.g., cyclohexamide, puromycin and rapamycin; (17) agents that affect microtubulin formation or stability such as, e.g., Vinblastine, Vincristine, 5 colchicine, 4-hydroxyphenylretinamide and paclitaxel; (18) agents that raise intracellular calcium levels by causing the release thereof from intracellular stores, such as, e.g., thapsigargin (Thastrup et al., Proc. NatL. Acad. Sci. U.S.A. 87:2466-2470, 1990), thapsigargicin (Santarius et al., Toxicon 25:389-399, 1987) and excitatory amino acids and their derivatives such as, e.g., kainate, N-methyl-D-aspartic acid (NMDA), N 10 acetylaspartylglutamate (NAAG, a glutamate derivative), 2-amino-3-(3-hydroxy-5 methylisoxazol-4-yl)propionic acid (AMPA) and 2-amino-3 -(3-hydroxy-5 phenylisoxazol-4-yl)propionic acid (APPA, an AMPA derivative); and agents that are added to isolated mitochondria, such as (19) MPT inducers, e.g., Bax protein (Jurgenmeier et al., Proc. NatL. Acad Sci. US.A. 95:4997-5002, 1998); and (20) 15 calcium and inorganic phosphate (Kroemer et al., Ann. Rev. Physiol. 60:619-642, 1998). In one aspect of the apoptosis assays, cells that are suspected of undergoing apoptosis may be examined for morphological, permeability or other changes that are indicative of an apoptotic state. For example by way of illustration and not limitation, apoptosis in many cell types may cause altered morphological 20 appearance such as plasma membrane blebbing, cell shape change, loss of substrate adhesion properties or other morphological changes that can be readily detected by those skilled in the art using light microscopy. As another example, cells undergoing apoptosis may exhibit fragmentation and disintegration of chromosomes, which may be apparent by microscopy and/or through the use of DNA specific or chromatin specific 25 dyes that are known in the art, including fluorescent dyes. Such cells may also exhibit altered membrane permeability properties as may be readily detected through the use of vital dyes (e.g., propidium iodide, trypan blue) or the detection of lactate dehydrogenase leakage into the extracellular milieu. Damage to DNA may also be assayed using electrophoretic techniques (see, for example, Morris et al., BioTechniques 26:282-289, WO 00/79274 PCTUSOO/17380 123 1999). These and other means for detecting apoptotic cells by morphologic, permeability and related changes will be apparent to those familiar with the art. In another aspect of the apoptosis assays, translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is 5 quantified by measuring outer leaflet binding by the PS-specific protein annexin (Martin et al, J. Exp. Med 182:1545-1556, 1995; Fadok et al., J. Immunol. 148:2207 2216, 1992.). In a preferred format, exteriorization of plasma membrane PS is assessed in 96-well plates using a labeled annexin derivative such as an annexin-fluorescein isothiocyanate conjugate (annexin-FITC, Oncogene Research Products, Cambridge, 10 MA). In another aspect of the apoptosis assays, quantification of the mitochondrial protein cytochrome c that has leaked out of mitochondria in apoptotic cells may provide an apoptosis indicator that can be readily determined (Liu et al., Cell 86:147-157, 1996). Such quantification of cytochrome c may be performed 15 spectrophotometrically, immunochemically or by other well established methods for detecting the presence of a specific protein. Release of cytochrome c from mitochondria in cells challenged with apoptotic stimuli (e.g., ionomycin, a well known calcium ionophore) can be followed by a variety of immunological methods. Matrix assisted laser desorption ionization time of flight mass (MALDI-TOF) spectrometry 20 coupled with affinity capture is particularly suitable for such analysis since apo cytochrome c and holo cytochrome c can be distinguished on the basis of their unique molecular weights. For example, the SELDI system (Ciphergen, Palo Alto, USA) may be utilized to follow the inhibition by mitochondria protecting agents of cytochrome c release from mitochondria in ionomycin treated cells. In this approach, a cytochrome c 25 specific antibody immobilized on a solid support is used to capture released cytochrome c present in a soluble cell extract. The captured protein is then encased in a matrix of an energy absorption molecule (EAM) and is desorbed from the solid support surface using pulsed laser excitation. The molecular weight of the protein is determined by its time of flight to the detector of the SELDI mass spectrometer.

WO 00/79274 PCT/USOO/17380 124 In another aspect of the apoptosis assays, induction of specific protease activity in a family of apoptosis-activated proteases known as the caspases (Thomberry and Lazebnik, Science 281:1312-1316, 1998) is measured, for example by determination of caspase-mediated cleavage of specifically recognized protein 5 substrates. These substrates may include, for example, poly-(ADP-ribose) polymerase (PARP) or other naturally occurring or synthetic peptides and proteins cleaved by caspases that are known in the art (see, e.g., Ellerby et al., J. Neurosci. 17:6165-6178, 1997). The labeled synthetic peptide Z-Tyr-Val-Ala-Asp-AFC, wherein "Z" indicates a benzoyl carbonyl moiety and AFC indicates 7-amino-4-trifluoromethylcoumarin (Kluck 10 et al., 1997 Science 275:1132-1136, 1997; Nicholson et al., Nature 376:37-43, 1995), is one such substrate. Another labeled synthetic peptide substrate for caspase-3 consists of two fluorescent proteins linked to each other via a peptide linker comprising the recognition/cleavage site for the protease (Xu et al., Nucleic Acids Res. 26:2034-2035, 1998). Other substrates include nuclear proteins such as UI-70 kDa and DNA-PKcs 15 (Rosen and Casciola-Rosen, J. Cell. Biochem. 64:50-454, 1997; Cohen, Biochem. J 326:1-16, 1997). In another aspect of the apoptosis assays, the ratio of living to dead cells, or the proportion of dead cells, in a population of cells exposed to an apoptogen is determined as a measure of the ultimate consequence of apoptosis. Living cells can be 20 distinguished from dead cells using any of a number of techniques known to those skilled in the art. By way of non-limiting example, vital dyes such as propidium iodide or trypan blue may be used to determine the proportion of dead cells in a population of cells that have been treated with an apoptogen and a compound according to the invention. 25 The person of ordinary skill in the art will readily appreciate that there may be other suitable techniques for quantifying apoptosis, and such techniques for purposes of determining the effects of mitochondria protecting agents on the induction and kinetics of apoptosis are within the scope of the assays disclosed here. Assay of Electron Transport Chain (ETC) Activity in Isolated 30 Mitochondria: As described above, mitochondria associated diseases may be WO 00/79274 PCTUSOO/17380 125 characterized by impaired mitochondrial respiratory activity that may be the direct or indirect consequence of elevated levels of reactive free radicals such as ROS. Accordingly, a mitochondria protecting agent for use in the methods provided by the instant invention may restore or prevent further deterioration of ETC activity in 5 mitochondria of individuals having mitochondria associated diseases. Assay methods for monitoring the enzymatic activities of mitochondrial ETC Complexes I, II, III, IV and ATP synthetase, and for monitoring oxygen consumption by mitochondria, are well known in the art. (See, e.g., Parker et al., Neurology 44:1090-1096, 1994; Miller et al, J. Neurochem. 67:1897-1907 1996.) It is within the scope of the methods provided by 10 the instant invention to identify a mitochondria protecting agent using such assays of mitochondrial function. Furthermore, mitochondrial function may be monitored by measuring the oxidation state of mitochondrial cytochrome c at 540 nm. As described above, oxidative damage that may arise in mitochondria associated diseases may include 15 damage to mitochondrial components such that cytochrome c oxidation state, by itself or in concert with other parameters of mitochondrial function including but not limited to mitochondrial oxygen consumption, may be an indicator of reactive free radical damage to mitochondrial components. Accordingly, the invention provides various assays designed to test the inhibition of such oxidative damage by mitochondria 20 protecting agents. The various forms such assays may take will be appreciated by those familiar with the art and is not intended to be limited by the disclosures herein, including in the Examples. For example by way of illustration and not limitation, Complex IV activity may be determined using commercially available cytochrome c that is fully 25 reduced via exposure to excess ascorbate. Cytochrome c oxidation may then be monitored spectrophotometrically at 540 nm using a stirred cuvette in which the ambient oxygen above the buffer is replaced with argon. Oxygen reduction in the cuvette may be concurrently monitored using a micro oxygen electrode with which those skilled in the art will be familiar, where such an electrode may be inserted into the 30 cuvette in a manner that preserves the argon atmosphere of the sample, for example WO 00/79274 PCTUSOO/17380 126 through a sealed rubber stopper. The reaction may be initiated by addition of a cell homogenate or, preferably a preparation of isolated mitochondria, via injection through the rubber stopper. This assay, or others based on similar principles, may permit correlation of mitochondrial respiratory activity with structural features of one or more 5 mitochondrial components. In the assay described here, for example, a defect in complex IV activity may be correlated with an enzyme recognition site. Tertiary Screening Assays Compounds that possess the desired activity profile in secondary in vitro assays are tested for in vivo efficacy in the rodent middle cerebral artery occlusion 10 (MCAO) model of transient focal ischemia that is reported to produce ischemia analogous to MCAO branch occlusion in humans (Longa et al., Stroke 1:84-91, 1989). Initially, test compounds are administered by a continuous intravenous infusion before and during the ischemia/reperfusion period to ensure the greatest chance for experimental success. Once efficacy is established, experiments are conducted in which 15 efficacy is assessed as a post-treatment using single and multiple drug administration regimens. The efficacy of the test compounds is directly assessed by measuring the reduction of neuronal loss in the infarcted brain region using techniques such as magnetic resonance imaging. Other additional endpoints are then measured, including reduction of brain lactate production as a consequence of the switch from aerobic to 20 anaerobic metabolism after oxygen deprivation, reduction in DNA, protein and lipid oxidation products. EXAMPLE 10 ET-BASED ASSAYS FOR MONITORING FUSION OF SUBCELLULAR COMPARTMENTS Assays utilizing energy transfer can be used to monitor the fusion of 25 subcellular compartments such as, e.g., organelles. For example, mitochondria undergo changes, including fission and fusion, and the latter process involves apparently coordinated rearrangements of internal elements (i.e., the inner membrane, cristae, etc.) (for a review, see Bereiter-Hahn and Voth, Microscopy Research and Technique 27:198-219, 1994). Such changes are believed to be important for various WO 00/79274 PCT/USOO/17380 127 developmental processes. In a variety of organisms including yeast such as C. cerevisiae, insects such as D. melanogaster, invertebrates such as C. elegans, and mammals such as H. sapiens, fusion of mitochondria is mediated by GTPase proteins generally known as "mitofusins" (see Hales et al., Cell 90:121-129, 1997; Hermann et 5 al., J. Cell. Biol. 143:359-373, 1998; and published PCT patent application WO 98/55618). Mutations in the fuzzy onions (fzo) gene, which encodes a mitofusin in D. melanogaster, impair spermatogenesis and renders male insects sterile (Hales et al., Cell 90:121-129, 1997). Accordingly, in certain embodiments the present invention provides a 10 method of identifying an agent that alters (i.e., increases or decreases in a statistically significant manner) the fusion of mitochondria by assaying, in the absence and presence of a candidate agent, a mitochondrial fusion event. Such an agent is identified by contacting a first sample comprising one or more mitochondria with an ET donor molecule and a second sample comprising one or more mitochondria with an ET 15 acceptor molecule, contacting the first and second samples with one another in the absence and presence of a candidate agent under conditions and for a time sufficient to permit mitochondrial fusion, exciting the ET donor to produce an excited ET donor molecule, detecting a signal generated by energy transfer from the ET donor to the ET acceptor and comparing the signal generated in the absence of the candidate agent to the 20 signal generated in the presence of the candidate agent. In those certain preferred embodiments wherein the invention is directed to a method for identifying an agent that alters mitochondrial fusion, neither the ET donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and the ET donor and the ET acceptor each localize independently of one another to the same 25 submitochondrial site or to acceptably adjacent submitochondrial sites as provided herein. Typically, based upon the teachings provided herein, a person having ordinary skill in the art can readily determine when a candidate agent alters mitochondrial fusion, for example, by detecting a statistically significant change in the ET signal generated in the presence of the agent relative to the ET signal generated in the absence of the agent. 30 As noted above, conditions permissive for mitochondrial fusion events are known in the WO 00/79274 PCT/USOO/17380 128 art, such that those having ordinary skill in the art can readily determine what are suitable conditions for conducting the instant assay method without undue experimentation. By way of illustration and not limitation, such conditions may include those that permit fusion of isolated mitochondria, which refers to mitochondria that 5 have been removed from the milieu in which they occur naturally; such conditions may also include those that permit at least one sample population of mitochondrial to undergo fusion within cells. It is desirable to develop novel antibiotics or pesticides that function by selectively inhibiting mitofusin activity in undesirable insects or eukaryotic parasites 10 but have minimal or no effect on the mitofusin of desirable insects or plants or on mammalian hosts including humans. It is also desirable to identify and characterize agents that stimulate or inhibit intracellular mitochondrial fusion events for the treatment of human diseases. The present invention can be used to achieve these goals in the following manner. 15 In general, a first group of mitochondria is preincubated with a donor compound, and a second group of mitochondria is incubated with an appropriate acceptor compound. Coincubation of the first and second group of mitochondria will result in fusion of individual mitochondria from each set, in which case the donor and acceptor compounds will achieve proximity to each other. Thus, mitochondrial fusion 20 will lead to energy transfer that can be measured according to the present disclosure. If an agent that stimulates or inhibits mitochondrial fusion is also added to these reactions, the degree of energy transfer and/or the rate at which energy transfer occurs will increase or decrease, respectively. Candidate agents having an effect on the activity or level of expression of mitofusin proteins can thus be screened for and characterized via 25 an ET-based assay. EXAMPLE 11 ET-BASED ASSAYS FOR MONITORING LOCALIZATION OF AGENTS TO SPECIFIC SUBCELLULAR SITES Assays utilizing energy transfer can be used to monitor the influx or 30 efflux of agents into a specific subcellular compartment within isolated organelles or WO 00/79274 PCT/USOO/17380 129 intact cells; in the latter case, such assays can be used to estimate pharmacokinetic properties of candidate therapeutic agents. For example, agents comprising tertramethylrhodamine (TMR) or related moieties have been described. For example, oligonucleotides that are 5'-end labeled with TMR are available from Genomyx Corp. 5 (Foster City, CA), and dideoxynucleotides conjugated to rhodamine or dichlororhodamine moieties are available from the Perkin-Elmer Corp. (Norwalk, CT). General methods for preparing conjugates comprising NAO- or JC-1-based moieties are described in published PCT patent application WO 98/17826. Mitochondrial uptake of such agents can be evaluated using the present invention as follows. 10 The uptake of agents comprising tertramethylrhodamine (TMR) or related moieties into mitochondria can be monitored by preincubating mitochondria or cells containing mitochondria with a donor compound such as NAO, MitoTracker@ Green FM or MitoFluorTM Green for a period of time, after which the TMR-conjugated agent of interest is added. If the agent is taken up by mitochondria, the TMR or TMR 15 like portion thereof will act as an acceptor for energy emitted from the donor compound. Uptake of the agent can thus be followed as a function of either decreasing emission from the donor or increasing emission from the TMR or TMR-like moiety. Similarly, the uptake of agents comprising NAO or NAO-like moieties into mitochondria can be monitored by preincubating mitochondria or cells containing 20 mitochondria with an acceptor compound such as TMRM, TMRE or rhodamine 123 for a period of time, after which the NAO-conjugated agent of interest is added. If the agent is taken up by mitochondria, the NAO or NAO-like portion thereof will act as a donor for energy emitted from the acceptor. Uptake of the agent can thus be followed as a function of either increasing emission from the acceptor compound or decreasing 25 emission from the NAO or NAO-like moiety. Uptake of agents comprising JC- 1-based moieties are monitored in like fashion, except that donor or acceptor compounds appropriate for JC-1 and mitochondria (see Tables 2 and 3) are used.

WO 00/79274 PCT/USOO/17380 130 EXAMPLE 12 FRET-BASED ASSAY OF Ay IN PERMEABILIZED CELLS In the following experiments, FRET-based assays of Ay using NAO and TMR were carried out essentially as described in Examples 5-7, with the chief 5 exception being that the cells used in the experiments were permeabilized (unless otherwise indicated), typically by treatment with digitonin, although other permeabilizing agents may be used. A related exception is that, because the cells were permeabilized, it was not necessary to add an ionophore, (e.g., ionomycin), in order to facilitate the entry of calcium into cells. Instead, calcium was added to the media and 10 was free to enter the permeabilized cells in the absence of an ionophore. Control experiments were performed to demonstrate that the same response to different concentrations of calcium is seen in permeabilized cells in the presence or absence of ionomycin, which confirms that calcium ions freely enter the permeabilized cells to at least the same degree as ionomycin-treated cells. 15 Another noteworthy difference between the protocols of the assays described in this example and those described above is that cells not contacted with both the ET donor and ET acceptor compounds at the same time, as in Examples 5-7. Instead, cells were initially contacted with NAO only (0.04 uM; 5 minutes), rinsed 3 times at 37*C in HBSS buffer (although many other buffers are suitable for these 20 rinses), and then transferred to a plate reader, i.e., an instrument capable of reading the signal produced due to energy transfer in the assay, preferably an automated or semi automated instrument such as a FLIPRTM instrument, which is described above. Once in the plate reader, the signal due to NAO was monitored for about 1 minute before addition of TMR as the second member of the ET pair. As described 25 above and according to non-limiting theory, TMR accumulates in mitochondria as a function of Ay and quenches, via FRET, the fluorescent signal from NAO. As Ay changes in response to various compounds or conditions, the concentration of TMR in mitochondria changes in a corresponding manner, as reflected by changes in the signal corresponding to NAO quenching.

WO 00/79274 PCT/USOO/17380 131 Typically, the average of several readings during this interval was taken for analysis and is labeled "Q" in Figure 16. The quenching of NAO by TMR was monitored in real time to ensure that equilibrium was reached before the addition of test compounds and/or other agents. This steady state is labeled "R" in Figure 16. Cells 5 were washed and all reagents prepared in Hanks Balanced Salt Solution (HBSS) containing 20 mM HEPES buffer. Test compounds were then added and allowed to equilibrate for at least 5 minutes; the average of several readings that correspond to the latter interval is labeled as "S" in Figure 16. In the final phase of the assay (labeled "T" in Figure 16), Ay was collapsed by the direct addition of Ca2+ to permeabilized cells. 10 This step differs from the protocols of the preceding examples, wherein Ca2+-mediated changes in Ay were induced in intact (nonpermabilized) cells by an ionophore (e.g., ionomycin) that promoted entry of high concentrations of Ca2+ into the cytosol from the extracellular environment. In the protocol used in this example, TMR was present throughout the 15 assay after its addition thereto. This minimized potential leakage of TMR from the mitochondria after washing, and hence stabilized the baseline readings; this was not a feature of protocols described in the preceding examples wherein cells were washed to remove TMR before being placed into the plate reader. Another modification of the assay in this Example involved data 20 handling. The data were analyzed by dividing the signal recovered after Ca2+ addition (the difference of T-S) by the signal that was initially quenched by TMR (the difference of Q-R). These mathematical manipulations yielded a ratio different from those presented in Example 5 (i.e., "Ay-Dissipating Value" and "Efficacy Index"). Data such as those presented in Figures 16 and 17 can be used to derive a "Ratio of Recovery of 25 Initial Quenching" (hereafter, "RRIQ") according to the following formula: RRIQ = [ T-S ] / [ Q-R ] Agents that inhibit or block Ay collapse have an RRIQ that is less than 30 that of control samples exposed to Ca2+ but not treated with such agents. Conversely, WO 00/79274 PCT/USOO/17380 132 compounds that induce or enhance Ay collapse have an RRIQ that is greater than that of untreated controls. It is also possible to evaluate the direct effect of compounds on AY by examination of the values corresponding to the difference between R and S (i.e., R-S). 5 Hyperpolarizing agents such as oligomycin increase TMR uptake, and hence increase NAO quenching, which results in a positive value for R-S. Conversely, compounds that directly dissipate Ay yield a negative value for R-S. Validation studies indicated that Ca2+ showed a dose-dependent response, where high Ca2+ levels caused Ay to collapse to the initial levels with only 10 minimal recovery of Ay. However, intermediate Ca2+ levels permitted some recovery of Ay, which is often lost as mitochondria undergo secondary permeability transition. That this is indeed secondary transition was supported by the observation that such transition was inhibited by cyclosporin A and bongkrekic acid. In many cases, data from such situations were best analyzed by summing the area under the curve (AUC) 15 for a specific duration after Ca2+ addition, rather than by using an average response for a specified interval. In addition to the ability to monitor secondary Ay collapse due to permeability transition, the protocol used in the instant Example also permits identification of agents that influence mitochondrial Ca2+ uptake, such as RU-360. In 20 this case, the initial depolarization upon Ca2+ addition was diminished compared to untreated controls. Analysis was accomplished by comparing the maximum peak height immediately after Ca2+ addition. As shown in Figure 17, permeabilized cells treated with calcium alone (i.e., with no ionophore present) underwent a concentration-dependent response to 25 calcium ions, leading to a collapse of Ay at 100 ptM Ca 2 that was roughly equivalent, in terms of both the extent of response and time course, to that seen in cells treated with the Ay-collapsing agent CCCP. The data shown in Figures 16 and 17 were generated using SH-SY5Y cells permeabilized by digitonin (0.008% v/v) obtained from Sigma (St. Louis, MO). The cells were grown in media comprising 125 mM KCl, 2mM WO 00/79274 PCTUSOO/17380 133

K

2

HPO

4 , 5 mM HEPES, 4 mM MgCl 2 , 1 mM malate, 1 mM succinate, 1 mM glutamate, 1 uM EGTA, pH 7.0. In Figure 18, the RRIQs derived from the data shown in Figures 16 and 17 are plotted as a function of the concentration (log M) of calcium ions (Ca 2 +). This 5 Concentration Response Curve (hereafter, "CRC") yields an EC 50 of 56.5 tM for Ca 2 in permeabilized cells. Data presented in Example 3 demonstrated that, in intact (i.e., nonpermeabilized) cells, ruthenium red (an inhibitor of the mitochondrial calcium uniporter) modulated [ionomycin + Ca 2 ]-induced collapse of Ay (e.g., Figure 8). In 10 these experiments (the instant Example 12), by way of contrast, an ionophore (ionomycin) was used to facilitate the entry of Ca2 into the cytosol of cells. Figure 19 shows a CRC of RU-360 (concentrations from 0 to 25 nM), an inhibitor of the calcium uniporter that is more specific than ruthenium red, in digitonin-permeabilized cells treated with Ca 2 *. The results yielded an EC50 of 11 nM for RU-360 in permeabilized 15 and Ca 2 + treated cells. Unlike the results presented in Figures 18 and 20, wherein RRIQs are plotted as a function of the concentration of a candidate or control agent, the responses in Figure 19 were summarized by calculating the area under the respective curves (see Example 4 and Table 6) for each concentration of RU-360, rather than RRIQ values. 20 Data also presented in Example 3 show that in intact cells, cyclosporin A (hereafter, "CsA") modulated [ionomycin + Ca2+]-induced collapse of Ay (e.g., Figure 9) at a concentration of 10 pM. In digitonin-permeabilized cells treated with CsA, CsA moderated the Ca 2 +-induced Ay collapse with an EC 5 0 of 0.31 pM (Figure 20). The results presented in Figure 20 demonstrate an important advantage of using 25 permeabilized cells instead of intact cells for assays of this type: CsA has a relatively modest ability to enter intact cells, thus lessening its apparent intracellular activity of CsA, but freely enters permeabilized cells. The results presented in this Example demonstrate various useful embodiments of the assays of the invention that utilize permeabilized cells. According 30 to one such embodiment, a range of distinct permeabilization conditions is employed to WO 00/79274 PCTIUSOO/17380 134 determine those wherein the plasma membrane selective permeability of a cell is compromised while organellar membranes (e.g., one or both of the membranes surrounding mitochondria and/or chloroplasts) retain their selective permeability. Permeabilization conditions such as those described in the preceding 5 paragraph are desirable in certain screening assays designed to select or identify individual active compounds that influence the activity of certain organellar components from among a group of candidate agents, in order to achieve one or more of a variety of objectives, which may include: (1) to avoid the "false negatives", i.e., the failure to detect activity of candidate organelle-influencing agents that do not exhibit 10 activity in assays using intact (nonpermeabilized) cells due in whole or in part to their moderate or limited capacity to cross the plasma membrane; (2) to preferentially or exclusively select or identify active compounds that directly or indirectly effect the selective permeability of one or more membranes surrounding an organelle; (3) to allow for the concomitant contacting of mitochondria with two or more agents, wherein 15 each of such agents influences the activity of certain organellar components, and wherein each of such agents would otherwise require specific means to gain entry into cytosol. In the case of objective (3), the goal of the assay may be to select or identify, from a group of candidate agents, active compounds that are antagonists or agonists of agents that influence the activity of certain organelles and organellar components. In 20 the latter situation, permeabilization of cells allows one to contact organelles with (i) agents that influence the activity of certain organellar components and (ii) one or more candidate agents, with the desirable features of contacting organelles with both (i) and (ii) at the same time and with a minimum of manipulation of the cells used in the assay; such features are particularly useful in high throughput (HTS) assays. As an example of 25 the desirable aspect of achieving objective (3) in such assays, the results presented herein demonstrate the concomitant contacting of mitochondria in permeabilized cells with Ca 2 ', which otherwise requires the presence of an ionophore such as ionomycin to facilitate its entry into the cytosol, and bongkrekic acid, an anti-apoptotic agent that influences the activity of ANT (adenine nucleotide translocator), a protein that is 30 localized to a mitochondrial membrane. Within certain ranges of permeabilization WO 00/79274 PCT/USOO/17380 135 conditions, objectives (1), (2) and (3) can all be realized through one set of permeabilization conditions; such conditions are particularly useful in high throughput (HTS) assays wherein it is desired to investigate the effects of various combinations of two or more molecules known or suspected to influence the activity of certain 5 organelles and organellar components, optionally in further combination with one or more compounds known or suspected to influence (e.g., enhance or decrease or otherwise regulate the activity of) such molecules. The results presented above derived from cells exposed to calcium ions (Ca2) and RU-360, an inhibitor of the mitochondrial calcium uniporter (Figure 19) 10 illustrate an embodiment of the assays of the invention in which the permeabilization treatment is such that the plasma membrane has lost its capacity to maintain selective permeability, but intracellular (e.g., organellar) membranes remained selectively permeable. That is, under the appropriate permeabilization conditions, Ca2 diffused across a permeabilized plasma membrane into the cytosol, but Ca2 uptake into 15 mitochondria was inhibited by RU-360 in a concentration-dependent manner. Thus mitochondrial membranes were not permissive for CA2 diffusion and apparently through the activity of the calcium uniporter. EXAMPLE 13 FRET-BASED ASSAY OF Ay IN PERMEABILIZED CELLS 20 USING REDUCED ET MOLECULE CONCENTRATIONS In this example, FRET-based assays of Ay using NAO and TMR were conducted using permeabilized cells essentially as described in Example 12, except that reduced loading concentrations of ET donor and acceptor molecules were used, and the effects of four agents known to influence mitochondrial activity states were 25 demonstrated. According to non-limiting theory, the use of lower concentrations of the ET donor and acceptor molecules NAO and TMR avoids potential self-quenching by the potentiometric dye, and also avoids undesirable dissipation of AY as the cationic dye enters the mitochondrial matrix. FRET methods using digitonin-permeabilized SH-SY5Y cells were 30 essentially as described above in Example 12 and Figure 16, with exceptions as noted WO 00/79274 PCT/USOO/17380 136 herein. All reagents were from Sigma (St. Louis, MO) unless otherwise stated. Briefly, 96-well assay plates containing SY5Y cells were loaded with 85 nM NAO, washed and placed into the FLIPRTM instrument. Initial instrument readings were monitored to confirm sample integrity, as described above. Cells were next simultaneously 5 permeabilized with 0.01% digitonin and labeled with the ET molecule TMR (156 nM) and permitted to equilibrate, after which various concentrations of a mitochondrially active compound (oligomycin, bongkrekic acid, nigericin or ADP) were added and instrument readings at 5-second intervals collected as described above. After approximately 10-12 minutes either CCCP (0.5 pM) or Ca" (35 tM or 50 pM) was 10 added to collapse mitochondrial membrane potential and readings were taken for an additional 5-10 minutes. Figure 21 shows the superimposed FRET RFU time course plot obtained when various concentrations of oligomycin, a specific inhibitor of ATP synthase, was the added mitochondrially active compound. By way of non-limiting theory, 15 mitochondrial inner membrane hyperpolarization that was observed following exposure of permeabilized cells to oligomycin (Fig. 21A) resulted from inhibition of ATP synthase and the consequent inhibition of normal Ay dissipation via ADP phosphorylation. Increased quenching of NAO by TMR was thus observed in the presence of oligomycin in a dose-dependent manner between 5 and 12 minutes (EC50 = 20 0.25 tg/ ml). Fig. 21B shows a CRC generated by calculating R-S, as described above in Example 12, for each oligomycin concentration (data points are shown + SEM, R 2 0.4123). According to similar reasoning, exposure of permeabilized cells in the FRET assay to an excess of ADP should result in a transient loss of Ay as 25 mitochondrial membrane potential is dissipated by ATP synthase-mediated phosphorylation of ADP. Such a transient loss of Ay was observed when the added mitochondrially active compound was 6.25- 50 pM ADP (Fig. 22A, EC50 = 12.1 pM). Below 6.25 pM, the change in Ay was indistinguishable from control groups that were exposed to buffer alone (ANOVA F = 36.4). The CRC plot as a function of ADP 30 concentration is shown in Fig. 22B.

WO 00/79274 PCT/USOO/17380 137 Figure 23 shows the superimposed FRET RFU time course plot obtained when various concentrations of bongkrekic acid (BKA), a specific inhibitor of the mitochondrial adenine nucleotide translocase, was the added mitochondrially active compound. By way of non-limiting theory, mitochondrial inner membrane 5 hyperpolarization that was observed following exposure of permeabilized cells to BKA (Fig. 23A) resulted from inhibition of ADP entry into the mitochondrial matrix, and the consequent inhibition of normal Ay dissipation via ADP phosphorylation. BKA is also believed to forestall mitochondrial permeability transition resulting from Ca> load. Increased quenching of NAO by TMR was thus observed in the presence of BKA in a 10 dose-dependent manner between 5 and 11 minutes (EC50 = 0.09 pM). Following addition of Ca2 to the permeabilized cells, BKA potentiated the extent of Ay recovery and also moderated the secondary loss of Ay (Fig. 23A), due probably to permeability transition. Fig. 23B shows a CRC generated by calculating R-S, as described above in Example 12, for each BKA concentration (data points are shown + SEM, R 2 = 0.5822). 15 Figure 24 shows the superimposed FRET RFU time course plot obtained when various concentrations of nigericin, a specific potassium/proton exchanger that collapses the portion of the approximately 220 mV proton-motive force across the mitochondrial membrane that derives from a pH gradient, was the added mitochondrially active compound. By way of non-limiting theory, mitochondrial inner 20 membrane hyperpolarization that was observed following exposure of permeabilized cells to nigericin (Fig. 24A) resulted from compensation for the loss of the pH gradient by the intact mechanisms responsible for the electrochemical component of the mitochondrial membrane proton-motive force (e.g., electron transport). Increased quenching of NAO by TMR was thus observed in the presence of 0.09-0.75 tM 25 nigericin at timepoints between 6 and 12 minutes. At higher nigericin concentrations (e.g., > 3 pM), there was a steep loss of NAO quenching (Fig. 24A), presumably (and according to non-binding theory) due to inability of electron transport to compensate for ApH dissipation. Fig. 24B shows a bar graph generated by determining RFU, as described above in Example 12, for each nigericin concentration to determine the 30 concentrations at which Ay collapsed (> 1.5pM) and at which mitochondrial inner WO 00/79274 PCTUSOO/17380 138 membrane hyperpolarization was detectable (<0.75 piM) using the FRET assay conditions described herein (data points are shown + SE, ANOVA P <0.0005). From the foregoing, it will be appreciated that, although specific 5 embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A method for assaying mitochondrial membrane potential, comprising the steps of: (a) 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: (i) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule; and (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential.

2. The method of claim 1 wherein the excited energy donor molecule transfers energy to the energy acceptor molecule to produce an excited energy acceptor molecule, and the signal detected in step (c) results from energy released by the excited energy acceptor molecule. WO 00/79274 PCT/USOO/17380 140

3. The method of claim 1 wherein energy transfer from the first energy transfer molecule to the second energy transfer molecule results in a decrease in the detectable signal.

4. The method of claim 1, further comprising contacting the mitochondria with an agent that induces dissipation of mitochondrial membrane potential.

5. The method of claim 4 wherein the agent that induces dissipation of mitochondrial membrane potential is an ionophore.

6. The method of either claim 1 or claim 4, further comprising contacting the mitochondria with an agent that induces collapse of mitochondrial membrane potential.

7. The method of claim 6, wherein the agent that induces collapse of mitochondrial membrane potential is selected from the group consisting of CCCP and FCCP.

8. The method of claim 1 wherein the sample is washed prior to the step of detecting a signal.

9. The method of claim 1 wherein the signal detected in step (c) is compared with a reference signal.

10. The method of claim 9 wherein the reference signal is generated by an indicator selected from the group consisting of 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. WO 00/79274 PCT/USOO/17380 141

11. The method of claim 1 wherein the sample comprises one or more mitochondria that are present within at least one cell, and wherein the signal detected in step (c) is compared with a reference signal.

12. The method of claim 11 wherein the reference signal is generated from a subcellular site selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space, mitochondrial matrix, cytoplasm, nucleus, nuclear membrane and plasma membrane.

13. The method of claim 11 wherein the reference signal is generated from extracellular medium.

14. The method of claim I wherein mitochondria are present within at least one cell during at least one step.

15. The method of claim 14 wherein the cell is an organism.

16. The method of claim 14 wherein the cell is a cultured cell.

17. The method of claim 14 wherein the cell is a cybrid cell.

18. The method of claim 14, wherein the cell is a plant cell.

19. The method of claim 14 wherein the cell is an animal cell.

20. The method of claim 14 wherein the cell is present in a biological sample derived from a multicellular organism.

21. The method of claim 20 wherein the cell is a plant cell. WO 00/79274 PCT/USO0/17380 142

22. The method of claim 20, wherein the cell is an animal cell.

23. The method of claim 22, wherein the animal is a mammal.

24. The method of claim 23, wherein the mammal is a human.

25. The method of claim 24, wherein said human has, is suspected of having or is at risk of having a disease or disorder associated with organellar dysfunction.

26. The method of claim 25 wherein the organellar dysfunction is mitochondrial dysfunction.

27. The method of claim 25 wherein the organellar dysfunction is lysosomal dysfunction.

28. The method of claim 1 wherein the first energy transfer molecule localizes to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane and the second energy transfer molecule localizes to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane.

29. The method of claim 28 wherein the concentration of the first energy transfer molecule in the submitochondrial site does not change as a function of membrane potential, and the concentration of the second energy transfer molecule in the mitochondrial matrix decreases as a function of membrane potential.

30. The method of claim 29 wherein (a) the first energy transfer molecule has an excitation maximum at a wavelength of from about 373 nm to about 390 nm, and an emission maximum at a wavelength of from about 400 nm to about 500 nm; and WO 00/79274 PCT/USOO/17380 143 (b) the second energy transfer molecule has an excitation maximum at a wavelength of from about 400 nm to about 500 nm.

31. The method of claim 30 wherein (i) the first energy transfer molecule is a fusion protein, wherein said fusion protein comprises (a) a blue-shifted green fluorescent protein polypeptide having a mutation in at least one of Phe-64, Ser-65, Tyr-66, Val-68 and Tyr-145, and (b) a polypeptide sequence that localizes said fusion protein to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane; and (ii) the second energy transfer molecule is selected from the group consisting of DASPEI, DASPMI, 4-Di-1-ASP, 2-Di-1-ASP, DiOC 7 (3). DiOC 6 (3), JC-1 and SYTO@ 18 yeast mitochondrial stain.

32. The method of claim 29 wherein (a) the first energy transfer molecule has an excitation maximum at a wavelength of from about 425 nm to about 440 nm, and an emission maximum at a wavelength of from about 450 nm to about 535 nm; and (b) the second energy transfer molecule has an excitation maximum at a wavelength of from about 450 nm to about 530 nm.

33. The method of claim 32 wherein (i) said first energy transfer molecule is a fusion protein, wherein said fusion protein comprises (a) a cyan-shifted Green Fluorescent Protein polypeptide having a mutation in at least one of Phe-64, Ser-65, Tyr-66, Asn-146, Met-153, Val-163 and Asn-212, and (b) a polypeptide sequence that localizes said fusion protein to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane; and (ii) the second energy transfer molecule is selected from the group consisting of DASPEI, 2-Di-1-ASP, DiOC 6 (3), SYTO@ 18 yeast mitochondrial stain, rhodamine 6G, JC-1, NBD C6-ceramide and NBD C6-sphingomyelin. WO 00/79274 PCT/USOO/17380 144

34. The method of claim 29 wherein (a) said first energy transfer molecule has an excitation maximum at a wavelength of from about 470 nm to about 500 nm, and an emission maximum at a wavelength of from about 505 nm to about 565 nm; and (b) said second energy transfer molecule has an excitation maximum at a wavelength of from about 505 nm to about 565 nm.

35. The method of claim 34 wherein (i) said first energy transfer molecule is selected from the group consisting of nonylacridine orange, MitoTracker@ Green FM, MitoFluorTM Green, and a fusion protein, wherein said fusion protein comprises (a) a Green Fluorescent Protein polypeptide selected from the group consisting of a wildtype Green Fluorescent Protein polypeptide, a red-shifted Green Fluorescent Protein polypeptide having a mutation in one or more of Phe-64, Ser-65, Tyr-66, Gln-69, Ser-72 and Thr-203 and a yellow-shifted Green Fluorescent Protein polypeptide having a mutation in one or more of Phe-64. Ser-65, Tyr-66, Gln-69, Ser-72 and Thr-203, and (b) a polypeptide sequence that localizes said fusion protein to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane; and (ii) said second energy transfer molecule is selected from the group consisting of rhodamine 123, JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylrosamine and rhodamine B.

36. The method of claim 29 wherein (a) said first energy transfer molecule has an excitation maximum at a wavelength of from about 545 to about 560 nm, and an emission maximum at a wavelength of from about 565 to about 625 nm; and (b) said second energy transfer molecule has an excitation maximum at a wavelength of from about 565 to about 625 nm. WO 00/79274 PCTUSOO/17380 145

37. The method of claim 36 wherein (i) said first energy transfer molecule is MitoTracker@ Orange CMTMRos; and (ii) said second energy transfer molecule is DiOC 2 (5).

38. The method of claim 29 wherein (a) said first energy transfer molecule has an excitation maximum at a wavelength of from about 495 to about 510 nm, and an emission maximum at a wavelength of from about 510 to about 570 nm; and (b) said second energy transfer molecule has an excitation maximum at a wavelength of from about 510 to about 560 nm.

39. The method of claim 38 wherein (i) said first energy transfer molecule is a fusion protein, wherein said fusion protein comprises (a) a polypeptide sequence selected from the group consisting of a FLASH protein sequence and a yellow-shifted Green Fluorescent Protein polypeptide sequence having a mutation in one or more of Ser-65, Tyr-66, Ser-72 and Thr-203, and (b) a polypeptide sequence that localizes said fusion protein to a submitochondrial site selected from the group consisting of mitochondrial matrix and mitochondrial inner membrane; and (ii) said second energy transfer molecule is selected from the group consisting of JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylrosamine, rhodamine B and 4-dimethylamino-tetramethylrosamine.

40. The method of claim 1 wherein a relative amount of the signal generated by energy transfer is detected.

41. The method of claim 1 wherein the signal is detected over a period of time and a rate of change in the signal level is determined. WO 00/79274 PCT/USOO/17380 146

42. The method of claim 1 wherein the signal is detected over a period of time and integrated.

43. The method of claim 1 wherein membrane potential comprises an electric potential, a pH potential, or both.

44. The method of claim 1 wherein the first and second energy transfer molecules localize to within from about 10 angstroms to about 100 angstroms of each other.

45. The method claim 1 wherein the first and second energy transfer molecules localize to within from about 10 angstroms to about 50 angstroms of each other.

46. The method claim 1 wherein the first and second energy transfer molecules localize to within from about 20 angstroms to about 50 angstroms of each other.

47. The method of any one of claims 1-46 wherein the signal is generated by fluorescence resonance energy transfer.

48. A method for identifying an agent that alters mitochondrial membrane potential, comprising the steps of: (a) contacting, in the absence and presence of a candidate agent, 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: (i) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and WO 00/79274 PCTUSOO/17380 147 (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule; (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential; and (d) comparing the signal generated in the absence of the candidate agent to the signal generated in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial membrane potential.

49. A method for identifying a regulator of an agent that alters mitochondrial membrane potential, comprising the steps of: (a) contacting, in the absence and presence of a candidate regulator, (1) an agent selected from the group consisting of an agent that alters mitochondrial membrane potential and an agent identified according to the method of claim 48 and (2) 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: (i) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule; WO 00/79274 PCT/USO0/17380 148 (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential; and (d) comparing the signal generated in the absence of the candidate regulator to the signal generated in the presence of the candidate regulator, and therefrom identifying a regulator of an agent that alters mitochondrial membrane potential.

50. The method of claim 49 wherein the regulator is an agonist of the agent that alters mitochondrial potential.

51. The method of claim 49 wherein the regulator is an antagonist of the agent that alters mitochondrial potential.

52. The method of claim 49 wherein the agent that alters mitochondrial membrane potential is an apoptogen.

53. The method of claim 49 wherein the agent that alters mitochondrial membrane potential is selected from the group consisting of thapsigargin, an ionophore and an excitatory amino acid or derivative thereof.

54. The method of claim 53 wherein the ionophore is selected from the group consisting of ionomycin and A23187.

55. The method of claim 53 wherein the excitatory amino acid or derivative thereof is selected from the group consisting of glutamate, NAAG, NMDA, AMPA, APPA and kainate.

56. A method for identifying an agent that preferentially alters mitochondrial membrane potential in mitochondria from a first biological source without WO 00/79274 PCTUSOO/17380 149 substantially altering mitochondrial membrane potential in mitochondria from a second biological source, comprising the steps of: (a) contacting, in the absence and presence of a candidate agent, each of a first and a second biological 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: (i) the first sample is derived from a first biological source and the second sample is derived from a second biological source that is distinct from the first biological source, (ii) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and (iii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule in the presence of each of said first and second samples; (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule in the presence of each of said first and second samples, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential; and (d) comparing the signal generated in the presence of each of said first and second samples in the absence of the candidate agent to the signal generated in the presence of each of said first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters mitochondrial membrane potential

57. The method of claim 56 wherein the first and second biological sources are distinct biological species. WO 00/79274 PCTUSOO/17380 150

58. The method of claim 56 wherein the first biological source is a mammal suspected of having, diagnosed as having or predisposed to having a disease, and the second biological source is a mammal that is not suspected of having and has not been diagnosed as having or predisposed to having said disease.

59. The method of claim 58 wherein the first biological source is a human and the second biological source is a human.

60. The method claim 58 wherein the disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease and type II diabetes.

61. A method for identifying an agent that preferentially alters mitochondrial membrane potential in mitochondria from a first biological sample without substantially altering mitochondrial membrane potential in mitochondria from a second biological sample, comprising the steps of: (a) contacting, in the absence and presence of a candidate agent, each of a first and a second biological 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: (i) the first sample is derived from a first tissue and the second sample is derived from a second tissue that is distinct from the first tissue, (ii) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and (iii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule in the presence of each of said first and second samples; WO 00/79274 PCT/USOO/17380 151 (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule in the presence of each of said first and second samples, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential; and (d) comparing the signal generated in the presence of each of said first and second samples in the absence of the candidate agent to the signal generated in the presence of each of said first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters mitochondrial membrane potential.

62. The method of claim 61 wherein the first tissue and the second tissues are derived from the same subject.

63. The method of claim 61 wherein the first and second tissues are each derived from a subject of the same species.

64. The method of claim 61 wherein the first and second tissues are derived from subjects of distinct species.

65. A method of detecting the fusion of a first mitochondrion and a second mitochondrion, comprising the steps of: (a) contacting a first sample comprising one or more mitochondria with a first energy transfer molecule that is not endogenous to the mitochondria; (b) contacting a second sample comprising one or more mitochondria with a second energy transfer molecule that is not endogenous to the mitochondria; wherein: (i) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and WO 00/79274 PCTIUSOO/17380 152 (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (c) contacting the first sample with the second sample under conditions and for a time sufficient to permit mitochondrial fusion; (d) exciting the energy donor molecule to produce an excited energy donor molecule; and (e) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, and therefrom determining fusion of the first mitochondrion and the second mitochondrion.

66. A method of identifying an agent that alters the fusion of mitochondria, comprising the steps of: (a) contacting a first sample comprising one or more mitochondria with a first energy transfer molecule that is not endogenous to the mitochondria; (b) contacting a second sample comprising one or more mitochondria with a second energy transfer molecule that is not endogenous to the mitochondria; wherein: (i) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (c) contacting, in the absence and presence of a candidate agent, the first sample with the second sample under conditions and for a time sufficient to permit mitochondrial fusion; (d) exciting the energy donor molecule to produce an excited energy donor molecule; WO 00/79274 PCT/USO0/17380 153 (e) detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule; and (f) comparing the signal detected in the absence of the candidate agent to the signal detected in the presence of the candidate agent, and therefrom identifying an agent that alters the fusion of the mitochondria.

67. The method of any one of claims 48, 49, 56, 61 or 66 wherein the agent increases mitochondrial membrane potential.

68. The method of any one of claims 48, 49, 56, 61 or 66 wherein the agent dissipates mitochondrial membrane potential.

69. The method of any one of claims 48, 49, 56, 61 or 66 wherein the agent collapses mitochondrial membrane potential.

70. The method of any one of claims 48, 49, 56, 61 or 66 wherein the agent alters an equilibrium distribution of at least one ionic species on either side of a cellular membrane.

71. The method of claim 70 wherein the ionic species is Ca2 and the cellular membrane is a mitochondrial membrane.

72. The method of claim 69 wherein the agent that collapses mitochondrial membrane potential is an apoptogen.

73. The method of claim 69 wherein the agent that collapses mitochondrial membrane potential interacts with an adenine nucleotide translocator. WO 00/79274 PCTUSOO/17380 154

74. The method of claim 73 wherein the agent that collapses mitochondrial membrane potential is selected from the group consisting of atractyloside, carboxyatractyloside, bongkrekic acid and isobongkrekic acid.

75. A reagent for measuring mitochondrial Ay, comprising a FRET donor molecule and a FRET acceptor molecule, wherein the accumulation of at least one of said molecules in mitochondria is dependent on Ay and the accumulation of the other of said molecules in mitochondria is independent of Ay.

76. The reagent of claim 75 wherein the molecule that accumulates in mitochondria independent of Ay is selected from the group consisting of NAO, MitoTracker@ Green FM, MitoFluorTM, DAPI, and a fusion protein comprising (a) a polypeptide selected from the group consisting of a red- shifted Green Fluorescent Protein polypeptide, a yellow-shifted Green Fluorescent Protein polypeptide and a "FLASH" polypeptide, and (b) a polypeptide sequence that localizes the fusion protein to the mitochondrial matrix or inner membrane.

77. The reagent of either claim 75 or 76 wherein the molecule that accumulates in mitochondria in a manner dependent on Ay is selected from the group consisting of TMRM, TMRE, rhodamine 123, ethidum bromide, 4-Di-1-ASP, 2-Di-1-ASP and DASPEI.

78. A kit comprising the reagent of claim 75 and ancillary reagents for measuring mitochondrial Ay.

79. A method for assaying cellular membrane potential, comprising the steps of: (a) contacting a sample comprising at least one cellular membrane, simultaneously or sequentially and in either order, with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein: WO 00/79274 PCT/USOO/17380 155 (i) the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and (ii) the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; (b) exciting the energy donor molecule to produce an excited energy donor molecule; and (c) 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 membrane site changes as a function of membrane potential.

80. The method of claim 79 wherein the first energy transfer molecule localizes to a first membrane site selected from the group consisting of mitochondria, endoplasmic reticulum, Golgi, lysosome and plasma membrane and the second energy transfer molecule localizes to the same membrane site or to an acceptably adjacent membrane site selected from the group consisting of mitochondria, endoplasmic reticulum, Golgi, lysosome and plasma membrane.

81. The method of claim 79 wherein the concentration of the first energy transfer molecule in the first membrane site does not change as a function of membrane potential, and the concentration of the second energy transfer molecule in the membrane site decreases as a function of membrane potential.

82. The method of claim 79 wherein (a) the first energy transfer molecule has an excitation maximum at a wavelength of from about 373 nm to about 390 nm, and an emission maximum at a wavelength of from about 400 nm to about 500 nm; and WO 00/79274 PCT/USOO/17380 156 (b) the second energy transfer molecule has an excitation maximum at a wavelength of from about 400 nm to about 500 nm.

83. The method of claim 79 wherein (a) the first energy transfer molecule has an excitation maximum at a wavelength of from about 425 nm to about 440 nm, and an emission maximum at a wavelength of from about 450 nm to about 535 nm; and (b) the second energy transfer molecule has an excitation maximum at a wavelength of from about 450 rm to about 530 nm.

84. The method of claim 79 wherein (a) said first energy transfer molecule has an excitation maximum at a wavelength of from about 470 nm to about 500 nm, and an emission maximum at a wavelength of from about 505 nm to about 565 nm; and (b) said second energy transfer molecule has an excitation maximum at a wavelength of from about 505 nm to about 565 nm.

85. The method of claim 79 wherein (a) said first energy transfer molecule has an excitation maximum at a wavelength of from about 545 to about 560 nm, and an emission maximum at a wavelength of from about 565 to about 625 nm; and (b) said second energy transfer molecule has an excitation maximum at a wavelength of from about 565 to about 625 nm.

86. A method for identifying an agent that alters a cellular membrane potential, comprising the steps of: (a) contacting, in the absence and presence of a candidate agent, a sample comprising one or more cellular membranes simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein: WO 00/79274 PCT/USOO/17380 157 (i) the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule; (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and (d) comparing the signal generated in the absence of the candidate agent to the signal generated in the presence of the candidate agent, and therefrom identifying an agent that alters cellular membrane potential.

87. A method for identifying a regulator of an agent that alters cellular membrane potential, comprising the steps of: (a) contacting, in the absence and presence of a candidate regulator, (1) an agent selected from the group consisting of an agent that alters a cellular membrane potential and an agent identified according to the method of claim 86 and (2) a sample comprising one or more cellular membranes simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein: (i) the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; WO 00/79274 PCTUSOO/17380 158 (b) exciting said energy donor molecule to produce an excited energy donor molecule; (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and (d) comparing the signal generated in the absence of the candidate regulator to the signal generated in the presence of the candidate regulator, and therefrom identifying a regulator of an agent that alters cellular membrane potential.

88. A method for identifying an agent that preferentially alters a cellular membrane potential in a membrane from a first biological source without substantially altering cellular membrane potential in a membrane from a second biological source, comprising the steps of: (a) contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more cellular membranes simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein: (i) the first sample is derived from a first biological source and the second sample is derived from a second biological source that is distinct from the first biological source, (ii) the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and (iii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule in the presence of each of said first and second samples; WO 00/79274 PCT/USOO/17380 159 (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule in the presence of each of said first and second samples, wherein the concentration of at least one of said energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and (d) comparing the signal generated in the presence of each of said first and second samples in the absence of the candidate agent to the signal generated in the presence of each of said first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters cellular membrane potential

89. A method for identifying an agent that preferentially alters a cellular membrane potential in a membrane from a first biological sample without substantially altering a cellular membrane potential in a membrane from a second biological sample, comprising the steps of: (a) contacting, in the absence and presence of a candidate agent, each of a first and a second biological sample comprising one or more cellular membranes simultaneously or sequentially and in either order with each of a first and a second energy transfer molecule that is not endogenous to the sample, wherein: (i) the first sample is derived from a first tissue and the second sample is derived from a second tissue that is distinct from the first tissue, (ii) the first and second energy transfer molecules each localize independently of one another to the same membrane site or to acceptably adjacent membrane sites such that at least one of the energy transfer molecules localizes to a cellular membrane that forms a subcellular compartment, and (iii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule in the presence of each of said first and second samples; (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule in the presence of each of said first WO 00/79274 PCT/USOO/17380 160 and second samples, wherein the concentration of at least one of said energy transfer molecules in the subcellular compartment changes as a function of membrane potential; and (d) comparing the signal generated in the presence of each of said first and second samples in the absence of the candidate agent to the signal generated in the presence of each of said first and second samples in the presence of the candidate agent, and therefrom identifying an agent that preferentially alters a cellular membrane potential.

90. A method for detecting a specific type of cell in a sample, comprising the steps of: (a) 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: (i) the first and second energy transfer molecules each localize independently of one another to the same subcellular site or to acceptably adjacent subcellular sites, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule; and (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein at least one of said energy transfer molecules preferentially accumulates in said specific type of cell; wherein said signal correlates with the presence of said specific type of cell in said sample.

91. The method of claim 90, further comprising the step of comparing the signal generated in said sample with the signal generated from a control sample lacking said specific type of cell.

92. The method of claim 90, wherein said specific type of cell is a cancer cell. WO 00/79274 PCT/USOO/17380 161

93. A method for identifying a Aym stabilizing agent, comprising the steps of: (a) contacting, in the absence and presence of a candidate Ay.m stabilizing agent, (1) an agent that alters Aym and (2) 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: (i) the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites, the sites being selected from the group consisting of mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space and mitochondrial matrix, and (ii) said first energy transfer molecule is an energy donor molecule and said second energy transfer molecule is an energy acceptor molecule; (b) exciting said energy donor molecule to produce an excited energy donor molecule; (c) detecting a signal generated by energy transfer from said first energy transfer molecule to said second energy transfer molecule, wherein the concentration of at least one of said energy transfer molecules in the mitochondria changes as a function of membrane potential; and (d) comparing the signal generated in the absence of the candidate Aym stabilizing agent, to the signal generated in the presence of the candidate Aym stabilizing agent, and therefrom identifying Aym stabilizing agent.

94. The method of claim 93, wherein said mitochondria are contained within cells.

95. The method of claim 94, wherein said agent that alters Aym is an agent that increases the level of cytosolic Ca2+. WO 00/79274 PCT/USOO/17380 162

96. The method of claim 95, wherein said agent that increases the level of cytosolic Ca2+ is selected from the group consisting of a calcium ionophore and thapsigargin.

97. The method of claim 95, wherein said cells comprise one or more types of glutamate receptors.

98. The method of claim 97, wherein said agent that increases the level of cytosolic Ca2+ is an excitatory amino acid or a derivative thereof.

99. The method of claim 98, wherein said excitatory amino acid or derivative thereof is selected from the group consisting of glutamate, NAAG, NMDA, AMPA, APPA and kainate.

100. A Aym stabilizing agent identified according to the method of claim 99.

101. A method of treating stroke comprising administering the Aym stabilizing agent of claim 100 to a patient in need thereof.

102. The method of claim 14 wherein said cell is a permeabilized cell.

103. The method of claim 20 wherein said cell is a permeabilized cell.

104. The method of claim 94 wherein said cells are permeabilized cells.

105. The reagent of claim 77 wherein the first FRET molecule that accumulates in mitochondria is formulated to dissolve to an extent necessary to saturate a population of cells in an aqueous solution with said first molecule within 0.01 to 2 minutes after being contacted therewith, and the second molecule that accumulates in mitochondria is formulated to dissolve to an extent necessary to saturate a population of cells in an aqueous WO 00/79274 PCT/USOO/17380 163 solution with said second molecule within 2.5 minutes to about 5 minutes after being contacted therewith.

106. The reagent of claim 77 wherein one of said molecules that accumulates in mitochondria is dissolved in an aqueous solution, and the other of said molecules that accumulates in mitochondria is present in solid form in said reagent.

107. The reagent of claim 106 wherein said molecule that accumulates in mitochondria and that is present in solid form in said reagent is formulated to dissolve to an extent necessary to saturate a population of cells in an aqueous solution with said second molecule within 0.01 minutes to about 5 minutes after being contacted therewith.