Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
  • G01N33/5079—Mitochondria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the invention relates generally to biological assays for detecting physiological conditions within cells. More specifically, the invention relates to monitoring molecular interactions in subcellular compartments based on energy transfer from a first compound (the energy transfer donor) to a second compound (the energy transfer acceptor).
• the cell is the basic unit of life and comprises a variety of subcellular compartments including, for example, the organelles.
• An organelle is a structural component of a cell that is physically separated, typically by one or more membranes, from other cellular components, and which carries out specialized cellular functions.
• Organelles and other subcellular compartments vary in terms of, inter alia, their composition and number in cells derived from different tissues, among normal and abnormal cells, and in cells derived from different species.
• organelles and other subcellular compartments, and macromolecules specifically associated therewith represent novel targets for the development of agents that specifically impact, respectively, a particular tissue within an animal, abnormal (diseased) but not normal (healthy) cells, or cells from an undesired species but not cells from a desirable species.
• members of the Bcl-2 family of proteins associate with the outer membranes of mitochondria and with other cellular membranes.
• the translocation of Bcl-2 proteins from one intracellular position to another occurs during apoptosis, a process by which some abnormal (e.g., pre- cancerous) cells are directed to undergo programmed cell death (PCD), thus eliminating their threat to their host organism.
• PCD programmed cell death
• Means for monitoring the accumulation of Bcl-2 proteins in various subcellular compartments, or their translocation from one intracellular location to another, would allow identification of agents designed to impact apoptosis, and to assay the effects of such agents in cells.
• cytoplasmic cellular hybrids comprising the nucleus of one cell type and organelles (mitochondria) from another cell type have been prepared.
• cytoplasmic cellular hybrids comprising the nucleus of one cell type and organelles (mitochondria) from another cell type have been prepared.
• cytoplasmic elements mitochondria
• 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.
• 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.
• One objective of the present invention is to provide methods and compositions for monitoring and assaying processes within subcellular compartments and macromolecules associated therewith.
• the invention may be used in a predicative, diagnostic or prognostic modality.
• Another objective of the present invention is to provide methods for screening for and identifying agents that impact organelles and other subcellular compartments in specific ways.
• agents are specific for undesirable abnormal cells, or for the cells of an undesirable parasites, they are expected to have remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease- impeditive effects on patients comprising such undesirable cells.
• the present invention fulfils these needs and realizes these and other objectives.
• Other advantages of the invention are apparent from the disclosure.
• the present invention is directed in part to methods and compositions for monitoring cellular processes, conditions and molecules using energy transfer (ET) techniques.
• 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.
• the invention provides a method for assaying mitochondrial membrane potential, comprising the steps of contacting a sample comprising one or more mitochondria, simultaneously or sequentially and in either order, with each of a first and a second energy transfer molecule that is not endogenous to the mitochondria, wherein the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites that are mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and wherein the first energy transfer molecule is an energy donor molecule and the second energy transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule; and detecting a signal generated by energy transfer from the first energy transfer molecule to the second energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes as a function of membrane potential.
• the excited energy donor molecule transfers energy to the energy acceptor molecule to produce an excited energy acceptor molecule, and the signal detected results from energy released by the excited energy acceptor molecule.
• energy transfer from the first energy transfer molecule to the second energy transfer molecule results in a decrease in the detectable signal.
• the method comprises contacting the mitochondria with an agent that induces dissipation of mitochondrial membrane potential.
• the agent that induces dissipation of mitochondrial membrane potential is an ionophore.
• the method comprises contacting the mitochondria with an agent that induces collapse of mitochondrial membrane potential.
• the agent that induces collapse of mitochondrial membrane potential is CCCP or FCCP.
• 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.
• the reference signal is generated by an indicator of cell number, an indicator of mitochondrial mass, an indicator of cellular protein, an indicator of cellular DNA, an indicator of mitochondrial DNA, an indicator of mitochondrial protein and an indicator of fluid volume.
• 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.
• the reference signal is generated from a subcellular site that may be a mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space, mitochondrial matrix, cytoplasm, nucleus, nuclear membrane or plasma membrane.
• the reference signal is generated from extracellular medium.
• 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, a plant cell or an animal cell.
• the cell is present in a 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.
• the human has, is suspected of having or is at risk of having a disease or disorder associated with organellar dysfunction, which in certain further embodiments is mitochondrial dysfunction and in certain other embodiments is lysosomal dysfunction.
• the first energy transfer molecule localizes to a submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane
• the second energy transfer molecule localizes to a submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane.
• concentration of the first energy transfer molecule in the submitochondrial site does not change as a function of membrane potential
• the concentration of the second energy transfer molecule in the mitochondrial matrix decreases as a function of membrane potential.
• 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 nm to about 500 nm.
• the first energy transfer molecule is a fusion protein, wherein the fusion protein comprises 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 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 mitochondrial stain.
• 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.
• 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 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-l-ASP, DiOC 6 (3), SYTO® 18 yeast mitochondrial stain, rhodamine 6G, JC-1, NBD C6-ceramide or NBD C6-sphingomyelin.
• 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
• the 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 the second energy transfer molecule has an excitation maximum at a wavelength of from about 505 nm to about 565 nm.
• the first energy transfer molecule is nonylacridine orange, MitoTracker® Green FM, MitoFluorTM Green or a fusion protein
• 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- 203, and a polypeptide sequence that localizes the fusion protein to a submitochondrial site that is mitochondrial matrix or mitochondrial inner membrane
• the second energy transfer molecule is rhodamine 123, JC-1, tetrabromorhodamine 123, rhodamine 6G,
• the 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 the second energy transfer molecule has an excitation maximum at a wavelength of from about 565 to about 625 nm.
• the first energy transfer molecule is MitoTracker® Orange CMTMRos; and the second energy transfer molecule is DiOC 2 (5).
• the 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 the second energy transfer molecule has an excitation maximum at a wavelength of from about 510 to about 560 nm.
• the first energy transfer molecule is a fusion protein, wherein the fusion protein comprises a polypeptide 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 6G, TMRM, TMRE, tetramethylrosamine, rhodamine B and 4-dimethylamino- tetramethylrosamine.
• a relative amount of the signal generated by energy transfer is detected.
• the signal is detected over a period of time and a rate of change in the signal level is determined, and in certain other embodiments the signal is detected over a period of time and integrated.
• membrane potential comprises an electric potential, a pH potential, or both.
• 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 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.
• the signal is generated by fluorescence resonance energy transfer.
• the present invention provides a method for identifying an agent that alters mitochondrial membrane potential, comprising the steps 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 adjacent submitochondrial sites, the sites being 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 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 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 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 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 transfer molecule to the second energy transfer molecule, wherein the concentration of at least one of the energy transfer molecules in the mitochondria changes
• the regulator is an agonist of the agent that alters mitochondrial potential, and in another embodiment the regulator is an antagonist of the agent that alters mitochondrial potential.
• the agent that alters mitochondrial membrane potential is an apoptogen.
• the agent that alters mitochondrial membrane potential is thapsigargin, an ionophore or an excitatory amino acid or derivative thereof.
• the ionophore is ionomycin or A23187.
• the excitatory amino acid or derivative thereof is glutamate, NAAG, NMDA, AMPA, APPA or kainate.
• the invention provides a method for identifying an agent that preferentially alters mitochondrial membrane potential in 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 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 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
• first and second biological sources are distinct biological species, and in another embodiment 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 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 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 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 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 transfer molecule is an energy acceptor molecule; exciting the energy donor molecule to produce an excited energy donor molecule
• 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 mitochondria; wherein the first and second energy transfer molecules each localize independently of one another to the same submitochondrial site or to acceptably adjacent submitochondrial sites that are mitochondrial outer membrane, mitochondrial inner membrane, mitochondrial intermembrane space or mitochondrial matrix, and the first energy transfer molecule is an energy donor molecule and the second energy 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 molecule;
• the agent increases mitochondrial membrane potential, in certain other embodiments the agent dissipates mitochondrial membrane potential, in certain other embodiments the agent 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.
• the ionic species is Ca and the cellular membrane is a mitochondrial membrane.
• the agent that collapses mitochondrial membrane potential is an apoptogen, and in certain other 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.
• the invention provides a reagent for measuring mitochondrial ⁇ , 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 ⁇ and the accumulation of the other of the molecules in mitochondria is independent of ⁇ .
• the molecule that accumulates in mitochondria independent of ⁇ is NAO, MitoTracker® Green FM, MitoFluorTM, DAPI, or a fusion protein comprising a polypeptide that is a red- shifted Green Fluorescent Protein 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.
• the molecule that accumulates in mitochondria in a manner dependent on ⁇ is TMRM, TMRE, rhodamine 123, ethidum bromide, 4-Di-l-ASP, 2-Di-l-ASP or DASPEI.
• the invention also provides, in certain embodiments, a kit comprising the reagent just described and ancillary reagents for measuring mitochondrial ⁇ .
• 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 site that is mitochondria, endoplasmic reticulum, Golgi, lysosome or plasma membrane.
• 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.
• 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 nm to about 500 nm.
• 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.
• the 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 the second energy transfer molecule has an excitation maximum at a wavelength of from about 505 nm to about 565 nm.
• the 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 the second energy transfer molecule has an excitation maximum at a wavelength of from about 565 to about 625 nm.
• the invention provides a method for identifying an agent that alters a cellular membrane potential, comprising the steps of 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 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 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 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
• 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 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 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 transfer molecules in the
• the invention provides 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 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 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 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
• the invention provides 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 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 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 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
• 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 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 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.
• the method 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.
• the specific type of cell is
• the invention provides a method for identifying a ⁇ m stabilizing agent, comprising the steps of contacting, in the absence and presence of a candidate ⁇ m stabilizing agent, an agent that alters ⁇ m 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 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 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
• the mitochondria are contained within cells, and in a further embodiment the agent that alters ⁇ m 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 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 ⁇ m stabilizing agent identified according to the method just described. In another embodiment, the invention provides a method of treating stroke comprising administering the ⁇ m stabilizing agent to a patient in need thereof.
• Figure 1 schematically depicts direct and indirect methods for measuring energy transfer. Symbols: “ ⁇ EX ,” peak excitation wavelength; “ ⁇ EM ,” peak emission wavelength; “e,” energy; open box, receptive filter setting; closed box, closed filter setting.
• FIG. 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; “D MX ,” donor compound localizing to the matrix; “A IM ,” acceptor compound localizing to the inner membrane; “D ⁇ S ,” donor compound localizing to the intermembrane space; "e,” energy.
• CS cytosolic space
• OM outer membrane
• IS intermembrane space
• IM inner membrane
• MX matrix
• D MX donor compound localizing to the matrix
• a IM acceptor compound localizing to the inner membrane
• D ⁇ S donor compound localizing to the intermembrane space
• energy energy.
• Figure 3 shows representative data from FRET-based assays of ⁇ m .
• Fig. 3A data from a Type I assay
• Fig. 3B data from a Type II assay.
• Figure 4 shows titration of an ET donor molecule (NAO) and an ET acceptor molecule (TMRM) in FRET assays of ⁇ m .
• NAO ET donor molecule
• TMRM ET acceptor molecule
• Figure 5 shows calibration of the concentrations of an ET donor molecule (NAO) and an ET acceptor molecule (TMRM) in FRET assays of ⁇ m .
• Figure 6 shows time-course data from a FRET assay of ⁇ m using NAO and TMRM alone and in combination.
• Figure 7 shows Type I FRET ⁇ m 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 ⁇ m assay of various agents. Symbols: “MO,” media (HBSS) only; “C,” CCCP; “I+RR,” ionomycin and ruthenium red.
• Figure 9 shows Type I FRET ⁇ m 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 ⁇ collapsing agent CCCP.
• Figure 11 shows Type II FRET ⁇ m assay. Symbols: "MO,” results from samples treated with media (HBSS) only; “4BA;” results from samples treated with the ⁇ m -dissipating agent 4-bromo-A23187; “C,” and arrow indicate time of CCCP addition to samples.
• Figure 12 is a dose response curve for a ⁇ -dissipating compound
• Figure 13 is a dose response curve for a compound (cyclosporin A) that protects mitochondria against a ⁇ -dissipating compound (ionomycin).
• Figure 14 shows a dose-response curve of three cell lines to the ⁇ m - 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 ⁇ at higher concentrations of Ca 2+ .
• Figure 17 shows the same data presented in Figure 16 wherein mean values are plotted without error bars.
• the response of permeabilized cells to CCCP, an agent known to induce ⁇ 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 ⁇ in a manner that is roughly equivalent, in terms of both the extent of response and time course, to that seen in cells treated with CCCP.
• Figure 18 is a concentration response curve (CRC) of Ca 2+ in permeabilized cells that was generated from the data presented in Figures 16 and 17.
• CRC concentration response curve
• Figure 19 is a CRC of RU-360, an inhibitor of the mitochondrial calcium uniporter, in permeabilized cells that were also contacted with Ca 2+ .
• Figure 20 is a CRC of cyclosporin A, an agent known to modulate Ca 2+ - induced ⁇ collapse, in permeabilized cells that were also contacted with Ca 2+ .
• 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.
• 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.
• the present invention pertains in part to the use of intermolecular energy transfer to monitor intracellular and intraorganellar conditions.
• the 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.
• appropriately paired energy transfer donor and acceptor molecules can be selected that accumulate at acceptably adjacent sites as provided herein, to generate detectable signals.
• ET energy transfer
• donor an energy-contributing "donor” molecule
• acceptor 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. 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.
• ET donor molecule and the ET acceptor molecule are molecules that are not 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 another for a particular type of energy transfer to occur.
• 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.
• detection of the 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.
• the invention provides a method for assaying a cellular membrane potential
• the invention provides a method for assaying mitochondrial membrane potential. 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 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.
• 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.
• the subject invention method can employ any suitable ET donor molecule and ET acceptor molecule that can function as a donor-acceptor pair.
• the method of the present invention may be used to identify an agent that alters a cellular membrane potential, or to identify a molecule that is a regulator of such an agent.
• the invention is directed to a method for assaying mitochondrial membrane potential, wherein neither the ET donor molecule 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.
• the ET donor molecule and the ET 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 acceptors, or phosphorescent donors with phosphorescent or fluorescent acceptors.
• Fluorescence refers to luminescence (emission of light) that is caused by the abso ⁇ tion 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.
• fluorescence occurs as certain 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 noticeable time after the incident radiation stops.
• “Chemiluminescence” refers to luminescence resulting from a chemical reaction
• bioluminescence refers to the emission of light from living organisms or cells, organelles or extracts derived therefrom.
• a detectable signal that is generated by energy transfer between ET donor and acceptor molecules results from fluorescence 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. 275:1-13, 1994).
• the energy transfer from an excited fluorophore (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 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).
• an excited fluorophore e.g., an ET donor molecule
• an absorber e.g., an ET acceptor molecule
• 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 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).
• FRET FRET to monitor specific intermolecular and/or intramolecular interactions that involve specific inter- and intramolecular recognition 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.
• intermolecular recognition events including associative and dissociative events, e.g., affinity and binding interactions
• ET donor and ET acceptor fluorophores are typically situated on two different molecules that are known or believed to enter into close association with each other.
• intramolecular interactions are measured, the ET donor and acceptor fluorophores are present on the same molecule.
• the present invention is based on the unexpected observation that energy transfer can occur between ET donor and ET acceptor 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.
• a common subcellular compartment for example, an organelle, a sub organellar site or other subcellular locale.
• the present invention can be used to monitor a variety of conditions or processes within, or associated with, such subcellular compartments.
• 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 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.
• 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.
• the biological sample contains one or more cellular membranes, including the plasma membrane and intracellular membrane bounded 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 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, transformed cell lines and the like.
• 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.
• permeabilization serves as an alternative to the use of a calcium ionophore.
• certain detectably labeled molecules such as certain of the ET donor and/or ET acceptor molecules provided herein, may 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.
• 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 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 themselves be used as drugs or as drug candidates.
• 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, such as ⁇ -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.
• permeabilizing cells Those skilled in the art are familiar with methods for permeabilizing cells and can readily determine without undue 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 permeabilization, and the like (see, e.g., Schulz, Methods Enzymol. 792:280-300, 1990).
• cells may be permeabilized using any of a variety of known techniques, including addition of permeabilizing agents such as bacterial toxins, for example, streptolysin O, Staphylococcus aureus ⁇ -toxin (a.k.a. ⁇ -hemolysin); other hemolytic agents such as saponin; or exposure to one or more detergents (e.g., digitonin, Triton X-100, NP-40, n-Octyl ⁇ -D-glucoside and the like) at concentrations below those used to lyse cells and solubilize membranes (i.e., below the critical micelle concentration).
• permeabilizing agents such as bacterial toxins, for example, streptolysin O, Staphylococcus aureus ⁇ -toxin (a.k.a. ⁇ -hemolysin); other hemolytic agents such as saponin; or exposure to one or more detergents (e.g., digitonin, Triton X
• 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 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).
• the subject or biological source 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 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 cofactors including ions, mediators, precursors, catabolites and the like.
• 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; 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.
• lysosomal storage defects such as the mucopolysaccaridoses, I-cell disease, Wolman disease and cholesteryl ester storage disease (e.g., Du et al., 1998 Mol
• a reference signal may be generated by a reference compound 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.
• 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.
• 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).
• the reference signal is generated by an indicator of the mitochondrial mass, the mitochondrial number or the mitochondrial volume present.
• 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 mitochondrial number are known in the art, and may include, for example, quantitative staining of a representative biological sample.
• 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 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).
• mitochondrion selective reagents such as fluorescent dyes that bind to mitochondrial molecular components (e.g., nonylacridine orange, MitoTrackersTM) or potentiometric dyes that accumulate in mitochondria as a function of mitochondrial inner membrane electrochemical potential (
• mitochondrial mass, volume and/or number may be quantified by morphometric analysis (e.g., Cruz-Orive et al., 1990 Am. J. Physiol. 258:L148; Schwerzmann et al., 1986 J. Cell Biol. 102:97).
• morphometric analysis e.g., Cruz-Orive et al., 1990 Am. J. Physiol. 258:L148; Schwerzmann et al., 1986 J. Cell Biol. 102:97.
• morphometric analysis e.g., Cruz-Orive et al., 1990 Am. J. Physiol. 258:L148; Schwerzmann et al., 1986 J. Cell Biol. 102:97.
• 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.
• 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).
• a reference signal may be generated by a 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.
• an indicator may permit improved quantitative precision by calibration/ normalization of sample volumes.
• Many compounds that are suitable for use as such 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.
• 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.
• 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 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.
• a signal may be detected over a period of time such that one or more behaviors of the signal may be analyzed as a function of time.
• 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.
• a 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).
• 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).
• an amount of a signal may be cumulatively determined over a discrete time interval, to provide a summed signal (e.g., an integrated signal).
• any of the methods provided by the invention can be modified so as to also include a reference signal that correlates with a reference parameter of interest 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 involve any signal that can be correlated with the desired reference parameter but which does not interfere with detection of the test/assay signal.
• 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 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, ⁇ ) can be read over the same time course.
• the reference signal and the test/assay signal e.g., FRET, ⁇
• the reference need not be read at the same time as the test/assay signal.
• 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.
• reference signals include the following.
• cellular protein including mitochondrial protein
• 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.
• PI propidium iodide
• PI propidium iodide
• peak excitation 536 nm
• peak emission 617 nm when bound to a nucleic acid
• 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, 526 nm; AO:RNA, peak excitation, 460 nm; peak emission, 650 nm).
• the SYTO stains 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., 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.
• the present invention provides diagnostic and prognostic methods, as 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 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 patient known to not have the disease or disorder.
• a cellular process or condition e.g., a cellular membrane potential such as mitochondrial membrane potential
• a biological sample comprising a cellular 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
• 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.
• biological samples from the patient are prepared and tested for their response 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.
• pharmacogenetic studies using the invention are employed to determine the correlations between different treatments and specific measurements generated by the invention.
• 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.
• 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. Cell. Biol.
• 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 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.
• agents that act in a species-specific manner are identified by the screening methods of the invention.
• 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 ET acceptor or the ET donor) is localized to a membrane that forms one border of that 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.
• 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 include methods for assaying mitochondrial membrane potential ( ⁇ ) or pH potential ( ⁇ pH), 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.
• 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 donor molecule) of the ET molecule pair.
• cellular membrane e.g., alters the membrane potential
• ET acceptor or donor molecule e.g., ET acceptor or donor molecule
• a change in the rate of energy transfer between the ET donor and acceptor molecules correlates with the association of a detectably labeled 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).
• a detectably labeled molecule e.g., labeled with either an ET donor or ET acceptor
• a labeled subcellular compartment e.g., labeled with either an ET acceptor or ET donor.
• paired ET molecules wherein each pair comprises an ET donor molecule and an ET acceptor molecule.
• ET donor molecules energy-donating compounds
• ET acceptor molecules energy-accepting compounds
• 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 ( ⁇ or ⁇ m), association of a particular intracellular molecule or 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 abso ⁇ tion spectrum of the ET acceptor molecule, so that energy transfer from the donor to the acceptor can occur.
• an ET donor compound has an emission peak wavelength (herein, " ⁇ D(em)”) that is within several nm of the excitation peak wavelength of the acceptor compound
• ⁇ A(ex) 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
• ET donor molecules or ET acceptor molecules being ⁇ 60 nm, ⁇ 50 nm, ⁇ 40 nm, ⁇ 30 nm, ⁇ 25 nm, ⁇ 20 nm, ⁇ 15 nm, ⁇ 10 nm, ⁇ 5 nm or ⁇ 1 nm.
• 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.
• certain donor-acceptor pairs may be suitable for ET methodologies as provided herein even where energy 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 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.
• energy 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 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.
• 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 pu ⁇ oses of determining whether a detectable FRET signal can be generated.
• 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.
• screening of 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 compartment in a contemplated biological sample to be used in the subject invention method.
• 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 compartment in a contemplated biological sample to be used in the subject invention method.
• 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
• ET donor and ET acceptor molecules may vary as a function of solution and sample conditions employed (e.g., solvent selected, solvent and ionic strength, pH, nature of the sample, etc.).
• 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 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.
• ET acceptor molecules and ET donor molecules can serve as ET acceptor molecules 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.
• 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, 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).
• 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 compounds preferentially accumulate in a water-based subcellular site or compartment. Some such compounds are known to preferentially accumulate at particular subcellular locations.
• a moiety that directs a compound to a subcellular location can be conjugated to a donor or acceptor moiety in order to generate a donor or acceptor compound capable of preferentially accumulating at the subcellular location of choice.
• 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 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.
• proteins that can serve as donor and acceptor compounds include fusion proteins comprising a "FLASH" (fluorescein arsenical helix binder) sequence (Griffin et al., Science 281:269-212, 1998), or an aequorin protein or a green fluorescent protein (Kendall et al., Trends in Biotechnology 16:216-224, 1998, and references cited therein).
• FLASH fluorescein arsenical helix binder
• green fluorescent protein 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 wavelengths of these GFP derivatives.
• an expression vector comprising nucleotide sequences appropriate for gene expression can be manipulated to comprise (1) a first nucleic acid encoding a GFP derivative or FLASH polypeptide and (2) a second nucleic acid encoding a peptide 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 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.
• a further criterion is that the donor and acceptor compounds should accumulate in the subcellular compartment at the same site, which will permit ET to take place, or at acceptably adjacent sites.
• acceptably adjacent it is meant that such sites are within close enough proximity for ET to occur.
• 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 preferably 40 A or less from each other, depending on the donor-acceptor pair of compounds.
• 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 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.
• 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 space; (iii) the intermembrane space and the inner membrane; and (iv) the inner membrane and the matrix, including factors within the matrix.
• 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. U.S.A. °5:6803-6808, 1993), to the intermembrane space using cytochrome c protein sequences (Mahajan et al., Nature Biotech. 16:541-552, 1998), and to the outer membrane of mitochondria using hexokinase (Sui et al., Arch. Biochem. Biophys.
• Aequorin fusion protein derivatives have been targeted to mitochondria using cytochrome c oxidase protein sequences (Pinton et al., Biofactors 5:243-253, 1998; Rizzuto et al., Nature 358:325- 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, 707:160-164, 1990), FO-ATPase subunit 9 (J. Biol. Chem. 277:25208- 25212, 1996), manganese superoxide dismutase (Balzan et al., Proc. Natl. Acad. Sci. U.S.A. 92:4219-4223, 1995), and P-450(SCC) (Kumamoto et al., J. Biochem., Tokyo, 105:12-18, 1989).
• 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. 2(55:19384-19391, 1993).
• Other targeting sequences such as those from the Rieske iron-sulfiur protein (Madueno et al., J. Biol. Chem. 2(59:17458-17463, 1994), direct fusion proteins across the thylakoid membrane.
• aequorin fusion protein derivatives have been targeted to the nucleus using nucleoplasmin protein sequences (Badminton et al., J. Biol. Chem. 277:31210-31214, 1997).
• aequorin fusion protein derivatives have been targeted to the endoplasmic reticulum using calreticulin protein sequences (Kendall et al., Biochem. Biophys. Res. Commun. 759:1008-1016, 1992).
• aequorin fusion protein derivatives have been targeted to the Golgi plasma membrane using galactosyltransferase, SNAP-25, connexin and 5-HT 1A -receptor protein sequences (Burton et al., Mol. Cell. Biol.
• GFP fusion proteins have been targeted to the Golgi apparatus using galactosyltransferase protein sequences (Llopis et al., Proc. Natl. Acad. Sci. U.S.A. 95:6803-6808, 1993)
• ET donor and acceptor molecules should occur preferentially at sites within the mitochondrion or whichever organelle or subcellular compartment is of interest.
• 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 specific to the organelle of interest can be followed despite accumulation(s) of compound(s) at secondary sites.
• 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 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 ⁇ D(ex), to samples; (2) the need to measure energy within the emission spectrum of the acceptor compound, preferably at or near ⁇ A(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.
• the spectra of energy being applied to samples to excite a donor compound, and the spectra of energy being emitted by an excited acceptor compound and measured in samples will determine, in general, what type of instrument will be used. For example, although ⁇ D(em) should not be identical to ⁇ A(em), the minimal acceptable amount of difference between these two values will be influenced by, among other factors, the instrumentation being used.
• ⁇ D(em) approaches ⁇ A(em)
• instruments capable of resolving closely-spaced wavelengths are required, and an assay using a donor-acceptor pair wherein the difference between ⁇ D(em) and ⁇ A(em) is less than about 3 to about 5 nm requires a high resolution instrument.
• an assay using a donor-acceptor pair wherein the difference between ⁇ D(em) and ⁇ A(em) is greater than about 50 to about 75 nm requires an instrument of medium to low resolution.
• a fluorometer is a device that measures fluorescent energy and should therefor be part of the instrumentation.
• a fluorometer 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., an fmaxTM fluorimetric plate reader, Molecular Devices Corp., Sunnyvale, CA; or a Cytofluor fluorimetric plate 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.
• a format such as, e.g., a 96-well microplate (such as, e.g., an fmaxTM fluorimetric plate reader, Molecular Devices Corp., Sunnyvale, CA; or a Cytofluor fluorimetric plate reader
• 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.
• an instrument capable of reading fluorescent signals in glass or polymeric 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 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. 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.
• FLIPRTM Fluorometric Imaging Plate Reader
• the FLIPRTM 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 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 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
• 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 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 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.
• 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.
• Mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical 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.
• ETC electron transport chain
• ATP adenosine triphosphate
• mitochondria 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 5(5:147-157). Apoptosis is apparently required for normal development of the nervous system and functioning of the immune system. Moreover, some disease 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).
• PCD programmed cell death
• agents that affect apoptotic events might have a variety of remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive uses.
• apoptogens are known to those familiar with the art (see, 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- ⁇ ); Fas ligand; glutamate; N-mefhyl-D-aspartate (NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et al., 1997 J. Cell. Biol.
• ionomycin and valinomycin ionomycin and valinomycin
• MAP kinase inducers such as, e.g.: anisomycin, anandamine
• cell 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., 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
• Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the 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.
• the cristae originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tubelike 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 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.
• ⁇ m 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. 91:221s and references cited therein).
• ⁇ m 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.
• ⁇ m is also the driving force for the influx of cytosolic Ca 2+ into the mitochondrion.
• ⁇ m collapses and mitochondrial membranes lose the ability to maintain an equilibrium distribution of one or more ionic species or other solutes, t.e., to selectively regulate permeability to solutes small (e.g., ionic Ca 2+ , Na", K + , H + ) and/or large (e.g., proteins).
• 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 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.
• MIMD mitochondrial diabetes and deafness
• 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. Assays for Measuring Changes in Parameters in Subcellular Compartments
• 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 compartment must be independent of the chosen parameter or activity.
• TMRM Fluorescence-Activated Reduction Reduction Reduction Reduction ⁇
• TMRE Error-Reduction Reduction ⁇
• rhodamine 123 Scaduto et al., Biophys. J. 76:469-411, 1999
• ethidium bromide Coppey-Moisan et al., Biophys. J.
• DASPMI 4-Di-l-ASP and 2- Di-l-ASP
• DASPEI DASPEI
• Compounds whose mitochondrial concentration is not dependent on ⁇ include, by way of example and not limitation, NAO (Maftah et al., Biophys. Res. Commun. 7(5 :185-190, 1989), MitoTracker® Green FM and MitoFluorTM Green (Haugland, Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., Molecular Probes, Inc., Eugene, OR, 1996, p.
• ⁇ collapse refers to the rapid dissolution of ⁇ , i.e., ⁇ reaches zero within a few minutes after mitochondria are treated with an agent that induces collapse of mitochondrial membrane potential, such as, for instance CCCP or FCCP or any other agent capable of rapidly driving ⁇ m to zero.
• ⁇ dissipation refers to a slower decrease in ⁇ that does not result in ⁇ 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 membrane potential, such as, for example, ionomycin, thapsigargin, atractyloside, 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 elevated intracellular cytosolic free calcium concentration.
• ETC mitochondrial electron transport chain
• mitochondria 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 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 257:1309-1312, 1998).
• MPT mitochondrial pore transition
• PCD programmed cell death
• proteins belonging to class (1) include hexokinase II, and Bcl-2, Bcl-X L , Bax and other members of the bcl-2 gene family (Kroemer, Nature Med.
• 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 275:1 132-1136, 1997), procaspase-2 and -9 (Susin et al., J. Exp. Med. 759:381-394, 1998) and apoptosis inducing factor (AIF; Susin et al., J. Exp. Med.
• Nucleic acids comprising nucleotide 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.
• aequorin or green fluorescent proteins such as wildtype GFP, BFP, CFP, RFP and YFP
• fluorescent derivatives that exhibit the same transient associations with mitochondria, or releases from mitochondria, as the corresponding parent proteins.
• hexokinase II fusion proteins that associate with the outer membrane of mitochondria (Sui et al., Arch. Biochem. Biophys.
• FLASH, aequorin and green fluorescent fusion proteins are used as donor or acceptor compounds in FRET- 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 ⁇ m .
• methods that utilize a single potentiometric fluorophore i.e., a fluorophore that accumulates in mitochondria in a ⁇ m -dependent manner
• the fluorophores may require that the fluorophores be present at concentrations that are toxic when agents that impact ⁇ m are introduced (see, e.g., U.S. Patent No. 5,169,788).
• the ET-based assays of ⁇ m of the invention can be carried out using lower, non-toxic doses of fluorophores.
• plasma membrane potential contributes to the signal in assays where a single potentiometric fluorophore is used, 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.
• the reference signal may be the signal observed in mitochondria with a known ⁇ m, and one or more such references signals may be used.
• ⁇ m may be evaluated relative to a ⁇ m 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 ⁇ m, or relative to a ⁇ m in a different type of mitochondria (e.g., mitochondria derived from a distinct subject or biological source).
• Specific embodiments of the present invention may employ different reference signals, as described in more detail below.
• 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 organisms as well. The reason for this is twofold: photosynthesis "fixes" atmospheric CO 2 into biologically usable carbohydrate (CHO) n molecules and also produces O 2 which is required by all aerobic organisms.
• chloroplasts Like mitochondria, chloroplasts have a double (outer and inner) 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 higher plants they are generally cylindrical in shape and range from about 5 to 10 ⁇ in length and from 0.5 to 2 ⁇ 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 of nuclear DNA (Jope et al., J. Cell. Biol. 79:631 -636, 1978).
• 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 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). 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 DNA, it appears that it is mainly ribosomal RNA (rRNA) molecules that are produced in the nucleolus.
• mRNA messenger
• rRNA ribosomal RNA
• 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 in the structure and/or composition of the lipid bilayer portion of the cell membrane can induce membrane curvature over a limited area thereof.
• 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 review, see Schekamn and Orci, Science 277:1526-1533, 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.
• the enzymes from the primary lysosome are brought into contact with, and are thus free to act upon, the contents of the endosome.
• its membrane is dissolved in order to release its contents into the cytosol.
• Cells are engineered to 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.
• an agent that is taken up in endosomes, wherein the agent is or has been modified to be an ET acceptor or donor, respectively.
• the acceptor and donor are present in the same subcellular compartment (the secondary lysosome), and ET occurs and can be monitored as described herein.
• 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.
• the endoplasmic reticulum is composed of a series of flattened 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.
• two types of ERs may exist in a cell.
• Smooth ER is generally tubular in shape and is typically devoid of attached ribosomes; one 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.
• the Golgi apparatus is a system of stacked, flattened and membrane- 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.
• organelles are also subcellular compartments within the scope of the invention.
• mitochondria, chloroplasts and nuclei 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 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.
• membrane-directed donors such as, e.g., 9-anthrylvinyl (LAPC)
• acceptors such as 3-perylenoyl (LPPC)
• the partition coefficients between membrane and aqueous phases are 8.3 x IO 3 and 10.5 x 10 D for LAPC and LPPC, respectively (Razinkov et al., Biochim. Biophys. Ada 7529:149-158, 1997).
• 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 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.”
• 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 “uncoating” and typically takes place in the cytoplasm (or a subcellular compartment, such as a vacuole, within the cytoplasm).
• 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 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.
• the donor can be 9-anthrylvinyl (LAPC) and the acceptor can be 3-perylenoyl (LPPC) (Razinkov et al., Biochim. Biophys. Acta 7529:149-158, 1997).
• LAPC 9-anthrylvinyl
• LPPC 3-perylenoyl
• Viral adsorption typically occurs equally well at 4°C and 37°C.
• uncoating proceeds rapidly at 37°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 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 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. tsutsugamushi typically grow only in the cytoplasm, organisms of the spotted fever 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 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.
• 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.
• 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 useful for treating Chlamydia infections.
• energy transfer is used to monitor interactions between pairs of macromolecules found within or associated with 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.
• 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 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 75:355-358, 1995; Kroemer, Nature Med. 5:614-620, 1997; WO95/13292; WO95/00160; and U.S. Pat. No. 5,015,568).
• Bcl-2 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 the interaction of Bcl-2 and Bax in individual mitochondria (Mahajan et al., Nat. Biotechnol. 16:541-552, 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.
• the width of the combined inner and outer mitochondria membranes has been estimated to be 22 + 4 nm (Perkins et al., J. Structural Biol. 779:260-272, 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 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.
• 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.
• the invention provides a method for identifying an 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.
• the subcellular compartment is a mitochondrion and the cellular membrane potential is 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 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 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.
• the invention is directed to a method for identifying an agent that preferentially alters mitochondrial membrane 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 sites as 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 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.
• an agent identified according to the instant method that is a species-specific agent or an agent that "preferentially" alters mitochondrial 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 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.
• 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 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.
• preferential alteration of mitochondrial membrane potential by such an agent may increase or may decrease ⁇ m , as long as the effect is species-specific.
• cells 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.
• an agent identified according to the instant method that that "preferentially" alters mitochondrial membrane potential in the 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 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.
• 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 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.
• preferential alteration of mitochondrial membrane potential by such an agent may increase or may decrease ⁇ m , as long as the effect is sample-specific.
• 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.
• the first and second tissues may be derived from distinct subjects of the same species, or from subjects of distinct species.
• an agent may be identified using this approach that preferentially alters neuronal cell mitochondrial membrane potential without substantially altering liver cell mitochondrial membrane potential.
• this embodiment of the invention may be used, for example, to identify agents that selectively induce collapse of ⁇ in mitochondria derived from different species, e.g., in trypanasomes (Ashkenazi et al., Science 257:1305-1308, 1998), and other eukaryotic pathogens and parasites, including but not limited to insects, but which do not induce ⁇ collapse in the mitochondria found in the cells of their mammalian hosts.
• agents are expected to be useful for the prophylactic or therapeutic management of such pathogens and parasites.
• members of the phylum Apicomplexa (formerly called Sporozoa) comprise a large and diverse group of pathogenic protozoa that are intracellular parasites.
• 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. 45:81-104, 1994, for a review).
• Antimalarial agents include agents that specifically impact the function of Plasmodium mitochondria (Peters et al., Ann. Trop. Med. Parsitol. 78:561- 579, 1984; Basco et al., J. Eukaryot. Microbiol. 47:179-183, 1994), and one such agent, atovaquone, collapses ⁇ in mitochondria from Plasmodium yoelii but has no effect on ⁇ of mammalian mitochondria (Srivastava et al., J. Biol. Chem. 272:3961-3966, 1997). Accordingly, the ET-based assay of ⁇ of the present invention can be used to screen libraries of compounds for novel antimalarial agents, i.e., compounds that cause ⁇ collapse in Plasmodium mitochondria but not in mammalian mitochondria.
• this embodiment of the invention is used to create and identify agents that selectively induce ⁇ collapse in mitochondria derived from 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.
• undesirable plants e.g., weeds
• desirable plants e.g., crops
• undesirable insects in particular, members of the family Lepidoptera and other crop- damaging insects
• desirable insects e.g., bees
• Cultured insect cells including for example, the Sf9 and Sf21 cell lines derived from Spodoptera frugiperda, and the HIGH FIVETM 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.
• the subcellular compartment of interest of a first species is loaded with a first donor-acceptor pair of molecules which fluoresce at a first wavelength
• the corresponding subcellular compartment from a second species is loaded with a second donor-acceptor pair of molecules which fluoresce at a second wavelength
• mitochondria from two different species may be loaded with such donor-acceptor pairs of molecules.
• the two types of 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.
• a Fluorometric Imaging Plate Reader (FLIPRTM) instrument may be used to rapidly alternate between 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.
• FLIPRTM Fluorometric Imaging Plate Reader
• 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.
• 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 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 agent that alters cellular membrane potential, and that in certain preferred embodiments alters mitochondrial membrane potential.
• 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 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 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 membrane potential or an agent that alters mitochondrial membrane potential and that is identified according to the methods provided herein.
• 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.
• 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 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. 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 and agents that collapse mitochondrial membrane potential (e.g., those described in 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 potential to alter mitochondrial membrane potential.
• 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.
• a regulator that is an agonist may potentiate such dissipation (e.g., cause collapse) while a 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.
• regulators that are agonists may also protect or enhance potential while regulators that are antagonists may lead to dissipation or collapse of ⁇ m .
• 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, including mitochondrial molecular components.
• intermolecular interaction events e.g., recognition, binding, complex formation, covalent modification, alteration of conformation
• an agent that alters mitochondrial membrane potential e.g., recognition, binding, complex formation, covalent modification, alteration of conformation
• mitochondrial molecular components are described, for example, in U.S. application number 09/161,172.
• the invention provides compositions and methods for monitoring mitochondrial membrane potential ( ⁇ ) and changes therein via energy transfer, as noted above.
• ⁇ mitochondrial membrane potential
• ⁇ is required for a variety of mitochondrial functions, and defects in the production or maintenance of ⁇ are associated with many diseases and disorders.
• changes in ⁇ occur in a variety of subcellular processes that can serve as targets for the development of therapeutic agents.
• the ET-based assay of ⁇ can be used to help confirm the presence of a disease or disorder associated with alterations in ⁇ 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) ⁇ and can thus be used to treat such diseases and disorders.
• the ET-based assay of ⁇ can be used to screen for agents that selectively perturb ⁇ in undesirable cells such as, 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. 97:227s-255s, 1981), including 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.
• ETC electron transport chain
• ATP adenosine triphosphate
• mitochondria are also involved in the genetically programmed cell suicide sequence known as "apoptosis" (Green and Reed, Science 281 :1309-1312, 1998; Susin et al., Biochim. et Biophys. Acta 7566: 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 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.
• ETC electron transport chain
• 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; diabetes mellitus, including Type I and Type II; mitochondria associated diseases, 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, MEL AS (mitochondrial encephalopathy, lactic acidosis, and stroke) and MIDD (mitochondrial diabetes and deafness); MERFF (myoclonic epilepsy ragged red fiber syndrome); arthritis; NARP (Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE (Myopathy and external ophthalmoplegia; Neuropathy
• ETC respiratory activity requires maintenance of an electrochemical potential ( ⁇ ) 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 "mitochondrial permeability transition" (MPT).
• MPT mitochondrial permeability transition
• 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).
• 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.
• ROS reactive oxygen species
• oxygen free radical induced lipid peroxidation is a well established pathogenetic mechanism in central nervous system (CNS) injury such as that found in a number of degenerative diseases, and in ischemia (i.e., stroke).
• CNS central nervous system
• 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.
• CNS neurodegenerative
• free radical mediated damage may inactivate one or more of the myriad proteins of the ETC.
• free radical mediated damage may result in catastrophic mitochondrial collapse that has been termed "transition permeability".
• transition 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, 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.
• permeability transition likely entails 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 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, gene products or related downstream mediator molecules and/or extramitochondrial 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 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. 502:1 178-1180, 1991; Reny, Int. J. Epidem. 25:886-890, 1994).
• Diabetes is a heterogenous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.
• 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 and increased levels of reactive oxygen species.
• 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 non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM).
• IGT insulin deficient non-insulin dependent diabetes
• diabetes mellitus NIDDM and IDDM
• NIDDM pancreatic beta cells
• end-organ response to insulin Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies, blindness and deafness.
• 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 significantly to the pathogenesis of diabetes mellitus (Anderson, Drug Dev. Res. 46:61- 79, 1999).
• mitochondrial tRNAs see, e.g., Suzuki et al., Diabetes Care 77:1428-1432, 1994; Kishimoto et al., Diabetologia 55:193-200, 1995; van der Ouweland et al., Muscle Nerve Suppl. 5:S124-S130, 1995; Hanna et al., Am. J.
• alterations in ⁇ may result in diabetic phenotypes in some instances, and individuals suspected of having or being predisposed to developing diabetes may be identified using the ET-based assay ⁇ of the invention.
• agents that increase and/or stabilize ⁇ 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 ⁇ 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 ⁇ is expected to be more likely to respond to agents that modulate ⁇ than a patient having mitochondria with a normal ⁇ . Parkinson's Disease
• Parkinson's disease 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. 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.
• MPTP neurotoxin l-mefhyl-4-phenyl-l,2,3,6- tetrahydropyridine
• 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 ⁇ might offer novel agents for preventing or 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 ⁇ of the invention.
• the ET-based ⁇ assay of the invention can be used to identify and characterize compounds that enhance or stabilize ⁇ , and these compounds 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, PD.
• the ET-based assay of ⁇ 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 ⁇ is expected to be more likely to respond to agents that modulate ⁇ than a PD patient having mitochondria with a normal ⁇ .
• AD Alzheimer's disease
• 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 et al., J. Exp. Med. 182:361-11, 1995; Zamzami et al., J. Exp. Med. 757:1661-72, 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.
• ROS reactive oxygen species
• AD Alzheimer's disease
• mitochondrial membrane potential In several cell types, including neurons, reduction in the mitochondrial membrane potential ( ⁇ ) 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). Moreover, cybrids comprising mitochondria derived from AD patients have lower resting mitochondrial membrane potentials than the corresponding parental SH-SY5Y cell line, and cyclosporin A reverses the depressed ⁇ in the AD cybrids (Cassarino et al., Biochem. Biophys. Res. Commun. 245:168-173, 1998).
• the ET-based assay ⁇ of the invention can be used to identify and characterize compounds that enhance or stabilize ⁇ , 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 ⁇ of the invention 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 ⁇ is expected to be more likely to respond to agents that modulate ⁇ than an AD patient having mitochondria with a normal ⁇ .
• mitochondrial dysfunction may be a critical factor in disease progression.
• ⁇ 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 ⁇ of the invention.
• the ET-based ⁇ assay of the invention can also be used to identify and characterize agents that enhance or stabilize ⁇ , and these 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, such disorders.
• the ET-based assay of ⁇ 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 ⁇ is expected to be more likely to respond to agents that modulate ⁇ than a patient having mitochondria with a normal ⁇ .
• 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.
• tissue ATP concentration drops to negligible levels within minutes.
• 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.
• ⁇ m 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 Chem 267:2934-2939, 1994; Zoratti et al., Biochim Biophys Ada 7247:139-176, 1995; Ellerby et al., J Neurosci 77:6165-6178, 1997). Consistent with these observations, glutamate-induced excitotoxicity can be inhibited by preventing mitochondrial Ca 2+ uptake or blocking MPT (Budd et al., J.
• such agents may be isolated by screening collections of compounds for their ability to stabilize ⁇ under excitotoxic conditions that mimic transient ischemia. Such agents are expected to have 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 ⁇ 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 ⁇ is expected to be more likely to respond to agents that modulate ⁇ than a patient having mitochondria with a normal ⁇ .
• 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 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-X L , block mitochondrial release of cytochrome c from mitochondria and the activation of caspase 3 (Yang et al, 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).
• overexpression of Bcl-2 and Bcl-X L protect against the mitochondrial dysfunction preceding nuclear apoptosis that is induced by chemotherapeutic agents.
• acquired multi-drug resistance to cytotoxic drugs is associated with inhibition of cytochrome c release that is dependent on overexpression of Bcl-X L (Kojima et al., J. Biol. Chem. 273: 16647- 16650, 1998).
• 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 ⁇ 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 ⁇ is expected to be more likely to respond to agents that modulate ⁇ than a patient having mitochondria with a normal ⁇ .
• the ET-based assay of mitochondrial ⁇ of the invention may also be used to identify agents that are selectively cytotoxic for hyperproliferative or other undesirable cell types.
• ⁇ is elevated in some carcinoma cell lines, and agents that accumulate in mitochondria as a function of ⁇ (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).
• the invention may be used to develop assays for subcellular conditions or intracellular processes, such as changes in mitochondrial ⁇ , in order to identify and characterize agents to treat degenerative disorders and diseases as well as hyperproliferative diseases.
• the ET-based assay of ⁇ 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.
• 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 the present invention.
• 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.
• concentrations of the donor and acceptor compounds at least two criteria apply.
• the concentrations of the donor and acceptor compounds should be sufficient for energy transfer to occur.
• 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 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.
• NAO is known to be toxic to certain cells at higher concentrations, for example at > 10 ⁇ M 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 of other toxic or protective factors are present, and the like.
• the neuroblastoma SH-S Y5 Y is a multiply subeloned 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. 26:1 191-1201, 1995; Pahlman et al., Ada Physiol. Scand. Suppl. 592:25- 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°) cells, and comprising mitochondria derived from patients having 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 inco ⁇ orated by reference).
• the 1685 cybrid cell line is one example of a cybrid cell line of this type.
• the 1685 cybrid cell line was created by fusing platelets from an AD donor with SH-SY5Y neuroblastoma cells that had been made rho° by extended treatment with ethidium bromide.
• 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 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 ⁇ and apoptotic events (see, e.g., Heerdt et al., Cancer Res. 59:1584-1591, 1999).
• a preferred cellular medium for MCF- 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 ⁇ g/ml bovine insulin).
• HBSS was generally used as cellul medium, but any media appropriate for a given cell line may be used in the assay.
• NAO stock solution was diluted 1 :5000 in Hank's balanced salt solution (HBSS, Life Technologies, Grand Island, NY) to yield a working stock solution containing 1 ⁇ g/ml NAO, which was further diluted as indicated below.
• HBSS Hank's balanced salt solution
• Probes, Inc., Eugene, OR; catalog #T668) was prepared in DMSO. This concentration 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 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.
• the FLIPRTM heaters and laser were turned on for at least 1 hour before 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.
• a centrally located 96-well microplate contains samples, and up to two 96-well plates, one on each side of the central plate, containing additional reagents can be included.
• 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 ⁇ collapsing agent (typically, CCCP), and the remaining Rows (C through H) contained the test compound(s) (e.g., candidate agents).
• a second reagent plate was set up so that each well contained an appropriate amount of a ⁇ collapsing agent (typically, CCCP) to be added to the samples sometime after the test compound(s).
• a ⁇ collapsing agent typically, CCCP
• test compounds Prior to the addition of test compounds, about 20 readings were taken on 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 ⁇ . For example, if cellular integrity was compromised, a significant collapse in ⁇ would be detected after the optional rinsing step but before addition of the test compounds. Next, the test compounds were added and 175 readings were taken at 5-second intervals.
• a ⁇ collapsing agent e.g., CCCP
• CCCP ⁇ collapsing agent
• 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 ( ⁇ F X ). Tests of significance for multiple (i.e., >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.
• >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.
• the initial instrument reading for each well was set to zero, and readings taken at 5-second intervals (following integrity confirmation as described above) numbered from about 21 to about 195-200 were summed ( ⁇ F X ).
• the readings during the final 4 minutes i.e., readings numbers about 214 to 230
• the ⁇ collapsing agent (CCCP) to maximally compromise membrane potential were averaged (F CCCP ). Because the use of ratios would violate mathematical assumptions inherent in ANOVA algorithms, the data were transformed (log or arcsin) before being evaluated for significance in one-way ANOVA analyses.
• the energy transfer from NAO to 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 [ ⁇ D(ex)], which in turn emits energy at a wavelength [ ⁇ D(em)] that overlaps the excitation spectrum of the acceptor, TMRM, and (b) measuring the emission from excited TMRM molecules at or near their peak emission wavelength [ ⁇ A(em)].
• NAO - TMRM FRET Indirect measurement of NAO - TMRM FRET also involves exciting NAO at ⁇ Dex, but it is the emission from the donor NAO, not from the acceptor TMRM, that is measured (i.e., ⁇ D(em) is measured rather than ⁇ A(em)). If energy transfer occurs efficiently from the excited donor (NAO) to the acceptor (TMRM), then emissions from the donor will be “quenched” and the signal at ⁇ D(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 ⁇ D(em) will increase).
• FRET was measured indirectly.
• TMRM + NAO loaded SY5Y cells were exposed to light of wavelength 488 nm (near ⁇ D(ex) for NAO, 485 nm) and the signal at 530 + 25 nm (near ⁇ D(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 from NAO (t.e., increasing fluorescence at or near ⁇ Dem).
• the signal at or near ⁇ D(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.
• 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.
• FRET occurred in wells E9, E10, F9, F10, 9G and 10G, as contrasted with the signals in wells A9 and AlO (NAO absent) and wells FI and F2 (TMRM absent).
• the signal in these wells may also include a significant background signal component derived from NAO alone (e.g., compare wells HI 1 and H12 to HI and H2) or TMRM alone (e.g., compare wells HI 1 and H12 to Al 1 and A12).
• NAO e.g., compare wells HI 1 and H12 to HI and H2
• TMRM e.g., compare wells HI 1 and H12 to Al 1 and A12
• useful preferred concentrations of NAO and TMRM for the assay include 50 ng/ml NAO and 10 ⁇ M TMRM (wells E9 and E10), 100 ng/ml NAO and 10 ⁇ M TMRM (wells F9 and F10), 200 ng/ml NAO and 10 ⁇ M TMRM (wells G9 and G10), and 200 ng/ml NAO and 5 ⁇ M TMR (wells G7 and G8).
• FRET was measured in cells treated with either 5 ⁇ M TMRM , 420 nM NAO, or with both compounds, for a more extended period after CCCP addition (1.5 ⁇ M).
• TMRM 5 ⁇ M TMRM
• NAO 5 ⁇ M nM NAO
• 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.
• NAO or TMRM was present alone, the fluorescent signal was essentially constant.
• 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 ⁇ than the control cybrid cells (MixCon) or the parental SH- SY5 Y cell line. This result demonstrates that the assay can be used to detect differences among cell types in reactions to agents that influence ⁇ .
• those that accumulate in mitochondria in a ⁇ -dependent manner include, by way of example and not limitation, rhodamine 123 (Emaus et al., Biochim. Biophys. Ada 550:436-448, 1986; Scaduto et al., Biophys. J. 76:469-477, 1999), TMRM and TMRE (Farkas et al., Biophys. J. 56:1053-1069, 1989; Ehrenberg et al., Biophys. J. 55:785-794, 1988).
• NAO specifically interacts with the inner mitochondrial membrane (Maftah et al., Biochem. Biophys. Res. Comm. 764:185-190, 1989).
• 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).
• TMRM, TMRE and rhodamine 123 localize to the inner mitochondrial 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).
• SH-SY5Y cells were cultured and assayed as in Example 1 with the following exceptions. Cells were incubated with an "acceptor” compound at 5 ⁇ M 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 ⁇ , CCCP, was added to the cells at a concentration of 1 ⁇ M, and relative fluorescence was measured using an fmaxTM (Molecular Devices, Inc., Sunnyvale, CA) fluorimetric plate reader (excitation, 485 nm; emission read at 538 nm + 20 nm).
• fmaxTM Molecular Devices, Inc., Sunnyvale, CA
• 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 ⁇ and exit of the acceptor compound (TMRM) from mitochondria. Because the donor compound (NAO) is retained by mitochondria independent of ⁇ , 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).
• energy transfer occurred only when the ET donor and acceptor molecules were appropriately co-localized within the subcellular compartment of interest.
• 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.
• a FRET-based assay designed to measure ⁇ of mitochondria as a model, a variety of agents are known in the art to lower (dissipate) or eliminate (collapse) ⁇ . Additionally, some agents are known to increase ⁇ above normal levels, i.e., to hyperpolarize mitochondria. Both types of agents were evaluated using the FRET-based assay of ⁇ .
• Oligomycin is an example of a compound that hyperpolarizes mitochondria.
• MixCon cybrid cells were contacted with TMRM (5 ⁇ M) and NAO (420 nM) as in Example 1.
• TMRM 5 ⁇ M
• NAO 420 nM
• a second set of MixCon cells was also treated with 10 ⁇ M 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 ⁇ collapse, was determined for eight separate wells of each of the three combinations of cells and agents using a FLIPRTM instrument.
• SH-SY5Y cells were treated with donor and acceptor compounds (respectively, NAO, 420 nm, and TMRM, 5 ⁇ M) according to the procedure described in Example 1, and HBBS media, CCCP (1.5 ⁇ M), ionomycin (5 ⁇ M), or ionomycin (5 ⁇ M) and BKA (2 ⁇ M; preincubated with cells at 37°C for 10 minutes before TMRM was added).
• RFU was monitored using a FLIPRTM instrument.
• 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 ⁇ and exodus from the mitochondria of the acceptor compound, TMRM.
• Treatment with ionomycin ultimately yielded a more gradual change in fluorescence, as was expected for an agent known in the art to cause a slower dissipation in ⁇ than CCCP.
• the addition of BKA to ionomycin-treated cells 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.
• Ionomycin and Ruthenium Red Ruthenium red was confirmed to have a protective effect with regard to the ⁇ -dissipating effects of ionomycin.
• Ionomycin is an ionophore that increases the level of cytosolic calcium; this leads to a dissipation of ⁇ as mitochondria take up 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 examples and treated with CCCP (1.5 ⁇ M), ionomycin (5 ⁇ M) with ruthenium red (100 ⁇ M) 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 ⁇ was essentially completely blocked by ruthenium red.
• cyclosporin A was confirmed to have a protective effect with regard to the ⁇ -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 TMRM as in the preceding examples, and treated with ionomycin (5 ⁇ M).
• One group of cells was preincubated with cyclosporin A (10 ⁇ M) for 15 minutes prior to CCCP addition. Fluorescence was measured over time at 530 + 25 nm using a FLIPRTM instrument.
• Atractyloside and Cyclosporin A The FRET assay described above and in the preceding examples was also validated by the fact that it showed a dissipation of ⁇ in SH-SY5Y cells treated with atractyloside (ATR, 5 mM) that peaked at about 6 minutes after ATR addition. At this concentration of ATR, ⁇ recovered after about 15 minutes, whereas CCCP (1 ⁇ M) led to a more complete collapse of ⁇ that was maintained for at least 15 minutes. Pretreatment with cyclosporin A (5 ⁇ M, 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.
• energy transfer occurred in a manner that accurately reflected changes in a parameter (in these examples, ⁇ ) 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 ⁇ ).
• agents known to increase e.g., oligomycin
• decrease e.g., CCCP. ionomycin, MPP , ATR
• protective agents BKA, ruthenium red, cyclosporin A
• the ET- based assay may be used to screen for and evaluate previously uncharacterized compounds for their effects on the chosen parameter (in this example, ⁇ ) and for their ability to counteract the effects of known compounds on the parameter of interest.
• 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 ⁇ (i.e., CCCP > ionomycin > ionomycin & BKA > media only). Thus, 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.
• Figure 3B shows a Type II assay.
• the symbols in Figure 3B are as follows.
• Optional initial readings (“A” or "B") that can be normalized to zero are first taken.
• the candidate ⁇ -dissipating compound is added at timepoint "2.” If the candidate ⁇ -dissipating compound has little or no effect on ⁇ , a signal like that represented by the solid line (“C") is expected, whereas a test compound that dissipates ⁇ results in a signal like that represented by the dashed line (“C").
• ⁇ -Dissipating activity of the test compound is calculated as the ⁇ -Dissipating Value according to the formula:
• the ⁇ -dissipating agent e.g., ionomycin, atractyloside, etc.
• the ⁇ -dissipating agent is added at timepoint "2." If the test compound has little or no effect on the activity of the ⁇ -dissipating agent, a signal like that represented by the dotted line ("C") is expected, whereas a test compound that inhibits or protects against the activity of the ⁇ -dissipating agent results in a signal like that represented by the solid line ("C ").
• an agent that completely collapses ⁇ e.g., CCCP
• D a reading
• the activity of the test compound is calculated as the Efficacy Index according to the formula:
• ⁇ collapsing agents include, by way of example and not limitation, valinomycin, A23187 and 4-Br-A23187.
• ⁇ collapsing agent it is desirable to establish a dose-response curve for whatever ⁇ collapsing agent is used, as conditions for the Type II assay are preferably such that ⁇ collapses, and the measured signal reaches a plateau, in a rapid manner (i.e., preferably within 5 minutes after addition of the ⁇ 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 ⁇ collapsing agent.
• a dose-response curve for CCCP is shown in Figure 10.
• SH-SY5Y cells were treated with 420 nM NAO and 5 ⁇ M TMRM according to the general procedure of Example 1 and then monitored for approximately 60 seconds before the indicated amount of CCCP 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.
• FIG. 11 shows relative fluorescence units + standard errors for readings taken at the indicated timepoints.
• SH-SY5Y cells were contacted with 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 ⁇ M of the ⁇ -dissipating agent 4-bromo-A23187. About 6.5 minutes later, the ⁇ -collapsing agent CCCP (final concentration, 5 ⁇ M) was added to all the samples and the fluorescence was read for an additional 7.5 minutes.
• CCCP final concentration, 5 ⁇ M
• the cells treated with 4-bromo-A23187 exhibited a gradual loss of ⁇ up until the time CCCP was added, at which point ⁇ further decreased and ultimately collapsed.
• the cells treated with media also showed a rapid loss of ⁇ following CCCP addition and approached complete ⁇ collapse, the MO and 4-BR curves becoming asymptotic after about 600 seconds and for the remainder of the experiment.
• TMRM and NAO were added at the concentrations and in the order and timing described in Example 1.
• ionomycin was added at various concentrations 10 minutes after addition of NAO.
• 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 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.
• Figure 13 the sum of the fluorescence signal over each time interval was determined and plotted against the log (10) ionomycin concentration (M) to generate a cyclosporin A dose-response curve.
• the dose response curves for cells exposed to ionomycin in three separate experiments 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 ⁇ .
• the FRET-based assay of ⁇ was performed on a neuroblastoma cell line (SH-SY5Y), and on the MixCon and 1685 cybrid cell lines that are generated from p° SY5Y cells.
• the control (MixCon) and Alzheimer's (1685) cybrids show the same general response to various agents and treatments that influence ⁇ , some differences were detected by the FRET-based assay.
• MixCon or 1685 cells (about 50,000 cells per well) were preincubated with 420 nM NAO and 5 ⁇ M TMRM according to the procedure of Example 1 , after which the calcium ionophore A23187 (0 to 5 ⁇ M) was added. Detectable loss of quenching of the NAO signal (i.e., fluorescence at 530 + 25 nm) was measured over 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 control (MixCon) cybrids were somewhat more sensitive to A23187 than the parental SH-SY5Y cells.
• Statistical analysis demonstrates that the increased susceptibility of the AD (1685) cybrid cells was significant.
• the ET-based assay of ⁇ 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 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.
• Assays utilizing energy transfer can be used to detect specific cell types in a biological sample.
• rhodamine 123 a Group II, III and IV acceptor compound; see Table 2
• rhodamine 123 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. 59:72-79, 1990).
• 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, MitoTracker® Green FM or MitoFluorTM 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.
• a mitochondrial donor compound from Group II, III or IV (Tables 2 and 3), such as NAO, MitoTracker® Green FM or MitoFluorTM Green
• NCI-H460 is a human lung large cell carcinoma cell line (see Example 1 for details). NCI-H460 cells were added to 96-well plates (about 50,000 cells per well). In a Type II ⁇ assay TMRM (5 ⁇ M) and NAO (420 nM) were added to the cells according to the procedure of Example 1.
• the ⁇ collapsing agent CCCP (5 ⁇ M) was added to all the samples about 9 minutes later. Fluorescence was measured using a FLIPRTM instrument during the experiment, as described above.
• differential susceptibility to inducers of ⁇ collapse can be used to distinguish cell types:
• 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.
• 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 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.
• ROS toxic reactive oxygen species
• glycolytic ATP production halts due to the lack of oxygen.
• glycolytic ATP production can continue under anoxic conditions, glycolysis cannot meet the energy demands of neurons due to limited stores of glycolysis substrates in the brain.
• 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 within minutes after cessation of oxygen flow to the brain.
• MPT membrane permeability transition
• healthy mitochondria play a bifunctional role in 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 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.
• the primary screening assay in stroke drug discovery utilizes the ET-based assay of ⁇ 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 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 ⁇ under control and Ca overload conditions.
• Glutamate receptors include ionotropic glutamate receptors (iGluRs) and metabotropic receptors (mGluRs).
• the iGluRs are glutamate-gated cation channels that 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, NMDAR1/2B, NMDAR1/2C and NMDAR1/2D.
• AMPA receptors are homomeric complexes including, for example, GluRl, GluR2, GluR3 and GluR4.
• Kainate receptors may be either homomeric or heteromeric complexes of 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 mGluR ⁇ .
• 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.
• extracellular glutamate elevates intracellular calcium levels (Stout et al., Nat. Neurosci. 7:366-373, 1998).
• changes in ⁇ are measured using the ET-based assay of ⁇ . Mitochondria-defective cybrid cells that have a depressed ⁇ (Cassarino et al., Biochem. Biophys. Res. Commun.
• 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 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 NMDAR1A/2B (Varney et al., J Pharmacol. Exp. Ther. 279:367-378, 1996); NMDAR2C, isoforms 1, 2, 3 and 4 (Dagget et al., J. Neurochem. 77:1953-1968, 1998); GluR3 (Varney et al., J. Pharmacol. Exp. Ther. 285:358-310, 1998); and GluRlb and GluR5a (Lin et al., Neuropharmacology 56:917-931, 1997).
• 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 sensitive indicator of the presence of oxidizing species.
• DCFC 2',7'- dichlorodihydroflurescein diacetate
• 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 back out of the cell.
• 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 (III)/ ascorbate and the relative mean DCFC fluorescence can be monitored as a function of time.
• 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 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.
• mitochondria protecting agents that scavenge free radicals stoichiometrically may not represent preferred agents because high intracellular concentrations of such agents might be required for them to be effective in vivo.
• mitochondria protecting agents that act catalytically may moderate production of oxygen radicals at their source, or may block ROS production 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 with the agent to reduce or prevent ROS generation.
• a mitochondria protecting agent 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 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.
• DASPMI Dimethylaminostyryl-N-Methylpyridinium
• DASPMI Upon introduction into cell cultures, DASPMI accumulates in 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 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.
• 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 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.
• physical e.g., UV radiation
• 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.
• apoptogens i.e., agents that induce apoptosis and/or associated processes when contacted with or withdrawn from cells or isolated mitochondria.
• apoptogens include by way of illustration and not limitation (1) apoptogens that are added to cells having specific receptors therefor, e.g., tumor 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.
• ionophores such as, e.g.: Staurosporine, Calphostin C, d-er tbro-sphingosine derivatives, Chelerythrine chloride, Genistein, l-(5-isoquinolinesulfonyl)-2-methylpiperazine, KN- 93, Quercitin, N-[2-((/?-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide and caffeic acid phenethyl ester; (7) ionophores such as, e.g.
• Ionomycin and valinomycin Ionomycin and valinomycin; (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, e.g.: cadmium; (15) DNA synthesis inhibitors such as, for example, Actinomycin D, 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, colchicine, 4-hydroxyphenylretinamide and
• thapsigargicin Santarius et al., Toxicon 25:389-399, 1987
• excitatory amino acids and their derivatives such as, e.g., kainate, N-methyl-D-aspartic acid (NMDA), N- 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.
• cells that are suspected of undergoing apoptosis may be examined for morphological, permeability or other changes that are indicative of an apoptotic state.
• apoptosis in many cell types may cause altered morphological 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.
• 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 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, 1999). These and other means for detecting apoptotic cells by morphologic, permeability and related changes will be apparent to those familiar with the art.
• vital dyes e.g., propidium iodide, trypan blue
• lactate dehydrogenase leakage into the extracellular milieu e.g., 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, 1999).
• translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is quantified by measuring outer leaflet binding by the PS-specific protein annexin (Martin et al, J. Exp. Med. 752:1545-1556, 1995; Fadok et al., J. Immunol. 745:2207- 2216, 1992.).
• 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, MA).
• 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 56:147-157, 1996).
• Such quantification of cytochrome c may be performed 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
• apoptotic stimuli e.g., ionomycin, a well known calcium ionophore
• Matrix- assisted laser desorption ionization time of flight mass (MALDI-TOF) spectrometry 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.
• 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.
• a cytochrome c 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.
• EAM energy absorption molecule
• the molecular weight of the protein is determined by its time of flight to the detector of the SELDI mass spectrometer.
• induction of specific protease activity in a family of apoptosis-activated proteases known as the caspases is measured, for example by determination of caspase-mediated cleavage of specifically recognized protein substrates.
• 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. 77:6165-6178, 1997).
• PARP poly-(ADP-ribose) polymerase
• the labeled synthetic peptide Z-Tyr-Val-Ala-Asp-AFC 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).
• substrates include nuclear proteins such as Ul-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, J. Cell. Biochem. 64:50-454, 1997; Cohen. Biochem. J. 526:1-16, 1997).
• 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 distinguished from dead cells using any of a number of techniques known to those skilled in the art.
• 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.
• mitochondria associated diseases may be 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 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 the instant invention to identify a mitochondria protecting agent using such assays of mitochondrial function.
• mitochondrial function may be monitored by measuring the oxidation state of mitochondrial cytochrome c at 540 nm.
• oxidative damage that may arise in mitochondria associated diseases may include 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.
• the invention provides various assays designed to test the inhibition of such oxidative damage by mitochondria 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.
• Complex IV activity may be determined using commercially available cytochrome c that is fully 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 cuvette in a manner that preserves the argon atmosphere of the sample, for example 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 mitochondrial components. In the assay described here, for example, a defect in complex IV activity may be correlated with an enzyme recognition site.
• test 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 (MCAO) model of transient focal ischemia that is reported to produce ischemia analogous to MCAO branch occlusion in humans (Longa et al., Stroke 7:84-91, 1989).
• MCAO rodent middle cerebral artery occlusion
• test compounds are administered by a continuous intravenous infusion before and during the ischemia/reperfusion period to ensure the greatest chance for experimental success.
• efficacy is established, experiments are conducted in which efficacy is assessed as a post-treatment using single and multiple drug administration regimens.
• test compounds 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 anaerobic metabolism after oxygen deprivation, reduction in DNA, protein and lipid oxidation products.
• Assays utilizing energy transfer can be used to monitor the fusion of subcellular compartments such as, e.g., organelles.
• subcellular compartments such as, e.g., organelles.
• 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 developmental processes.
• yeast such as C. cerevisiae
• insects such as D. melanogaster
• invertebrates such as C. elegans
• mammals such as H.
• GTPase proteins generally known as "mitofusins” (see Hales et al., Cell 90:121-129, 1997; Hermann et al., J. Cell. Biol. 745:359-373, 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).
• the present invention provides a 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 acceptor molecule, contacting the fust 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 signal generated in the presence of the candidate agent.
• 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.
• 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.
• conditions permissive for mitochondrial fusion events are known in the 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.
• such conditions may include those that permit fusion of isolated mitochondria, which refers to mitochondria that 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.
• 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.
• mitochondrial fusion 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 an ET-based assay.
• Assays utilizing energy transfer can be used to monitor the influx or efflux of agents into a specific subcellular compartment within isolated organelles or intact cells; in the latter case, such assays can be used to estimate pharmacokinetic properties of candidate therapeutic agents.
• agents comprising tertramethylrhodamine (TMR) or related moieties have been described.
• TMR tertramethylrhodamine
• oligonucleotides that are 5 '-end labeled with TMR are available from Genomyx Corp. (Foster City, CA), and dideoxynucleotides conjugated to rhodamine or dichlororhodamine moieties are available from the Perkin-Elmer Corp. (Norwalk, CT).
• TMR tertramethylrhodamine
• a donor compound such as NAO, MitoTracker® Green FM or MitoFluorTM Green
• the agent is taken up by mitochondria, the TMR or TMR- 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.
• the uptake of agents comprising NAO or NAO-like moieties into mitochondria can be monitored by preincubating mitochondria or cells containing 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 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.
• FRET-BASED ASSAY OF ⁇ IN PERMEABILIZED CELLS FRET-based assays of ⁇ using NAO and TMR were carried out essentially as described in Examples 5-7, with the chief 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 was free to enter the permeabilized cells in the absence of an ionophore.
• an ionophore e.g., ionomycin
• 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 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.
• 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.
• TMR TMR-induced oxidative stress
• FRET fluorescence resonance energy transfer spectrometry
• concentration of TMR in mitochondria changes in a corresponding manner, as reflected by changes in the signal corresponding to NAO quenching.
• Q 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.
• This step differs from the protocols of the preceding examples, wherein Ca -mediated changes in ⁇ were induced in intact (nonpermabilized) cells by an ionophore (e.g.,
• TMR was present throughout the 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.
• Agents that inhibit or block ⁇ collapse have an RRIQ that is less than
• permeabilized cells treated with calcium alone underwent a concentration-dependent response to calcium ions, leading to a collapse of ⁇ at 100 ⁇ M 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 ⁇ -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 KC1, 2mM 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.
• Example 3 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 ⁇ (e.g., Figure 8).
• an ionophore ionomycin
• 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+ .
• CsA modulated [ionomycin + Ca 2+ ]-induced collapse of ⁇ (e.g., Figure 9) at a concentration of 10 ⁇ M.
• CsA moderated the Ca 2+ -induced ⁇ collapse with an EC 50 of 0.31 ⁇ M ( Figure 20).
• Figure 20 The results presented in Figure 20 demonstrate an important advantage of using 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.
• results presented in this Example demonstrate various useful embodiments of the assays of the invention that utilize permeabilized cells.
• a range of distinct permeabilization conditions is employed to 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 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 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 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.
• 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.
• 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.
• HTS high throughput
• 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 localized to a mitochondrial membrane.
• an ionophore such as ionomycin
• 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 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.
• HTS high throughput
• FRET-based assays of ⁇ 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 demonstrated.
• 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 ⁇ as the cationic dye enters the mitochondrial matrix.
• FRET methods using digitonin-permeabilized SH-SY5Y cells were essentially as described above in Example 12 and Figure 16, with exceptions as noted 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 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 ⁇ M) or Ca 2+ (35 ⁇ M or 50 ⁇ M) was added to collapse mitochondrial membrane potential and readings were taken for an additional 5-10 minutes.
• a mitochondrially active compound oligomycin, bongkrekic acid, nigericin or ADP
• 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.
• FIG. 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.
• BKA bongkrekic acid
• mitochondrial inner membrane hyperpolarization that was observed following exposure of permeabilized cells to BKA (Fig. 23 A) resulted from inhibition of ADP entry into the mitochondrial matrix, and the consequent inhibition of normal ⁇ dissipation via ADP phosphorylation.
• BKA is also believed to forestall mitochondrial permeability transition resulting from Ca 2+ load.
• 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.
• mitochondrial inner 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).
• 24B shows a bar graph generated by determining RFU, as described above in Example 12, for each nigericin concentration to determine the concentrations at which ⁇ collapsed (> 1.5 ⁇ M) and at which mitochondrial inner membrane hyperpolarization was detectable ( ⁇ 0.75 ⁇ M) using the FRET assay conditions described herein (data points are shown + SE, ANOVA P O.0005).

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