CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
1
COMPOSITIONS AND METHODS FOR ASSAYING SUBCELLULAR
CONDITIONS AND PROCESSES USING ENERGY TRANSFER
TECHNICAL FIELD
The invention relates generally to biological assays for detecting
physiological conditions within cells. More specifically, the invention
relates to
monitoring molecular interactions in subcellular compartments based on energy
transfer
from a first compound (the energy transfer donor) to a second compound (the
energy
transfer acceptor).
BACKGROUND OF THE INVENTION
The cell is the basic unit of life and comprises a variety of subcellular
compartments including, for example, the organelles. An organelle is a
structural
component of a cell that is physically separated, typically by one or more
membranes,
from other cellular components, and which carries out specialized cellular
functions.
Organelles and other subcellular compartments vary in terms of, inter alia,
their
composition and number in cells derived from different tissues, among normal
and
abnormal cells, and in cells derived from different species. Accordingly,
organelles and
other subcellular compartments, and macromolecules specifically associated
therewith,
represent novel targets for the development of agents that specifically
impact,
respectively, a particular tissue within an animal, abnormal (diseased) but
not normal
(healthy) cells, or cells from an undesired species but not cells from a
desirable species.
For example, members of the Bcl-2 family of proteins (discussed in
more detail infra) associate with the outer membranes of mitochondria and with
other
cellular membranes. The translocation of Bcl-2 proteins from one intracellular
position
to another occurs during apoptosis, a process by which some abnormal (e.g.,
pre-
cancerous) cells are directed to undergo programmed cell death (PCD), thus
eliminating
their threat to their host organism. Means for monitoring the accumulation of
Bcl-2
proteins in various subcellular compartments, or their translocation from one
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
2
intracellular location to another, would allow identification of agents
designed to impact
apoptosis, and to assay the effects of such agents in cells.
As another example, cytoplasmic cellular hybrids (cybrids) comprising
the nucleus of one cell type and organelles (mitochondria) from another cell
type have
been prepared. Experiments with such cybrids have demonstrated that cellular
defects
associated with diseased cells are transferred with cytoplasmic elements
(mitochondria)
from diseased cells to cybrids. Diseases that have been demonstrated to have a
cytoplasmic component in this manner include Alzheimer's disease and
Parkinson's
disease (Swerdlow et al., Neurology -19:918-925, 1997; Swerdlow et al., Annals
of
Neurology -10:663-671, 1996). Means for monitoring intracellular processes
during the
formation of cybrids, or for comparing intracellular processes between cybrids
that have
a common nuclear background but that differ according to the sources of donor
cytoplasm as their sources of mitochondria, would allow one to study the
mechanisms
of such processes and to identify agents that impact such processes.
By way of further example, it is possible to develop antibacterial agents
by taking advantage of the fact that bacterial cells comprise structures
(e.g., cell walls)
that are not present in eukaryotic cells, and by developing agents that
specifically
impact these structures. In contrast, it has been more difficult to develop
agents to treat
diseases and disorders resulting from eukaryotic parasites of mammals
including
humans, in part because of the fact that many cellular features of such
parasites have
structural similarities to homologous structures found in the host's cells; as
a result, any
agent that negatively impacts a cellular component of such a parasite is also
likely to
have a negative effect on the analogous component of the eukaryotic host
cells.
There is thus a need for methods and compositions that allow for the
rapid and detailed monitoring of processes within subcellular compartments and
macromolecules associated therewith. Further, there is a need for methods and
compositions for identifying and screening for agents that impact such
processes in
specific instances.
One objective of the present invention is to provide methods and
compositions for monitoring and assaying processes within subcellular
compartments
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
3
and macromolecules associated therewith. When such processes are associated
with
particular diseases and/or disorders, the invention may be used in a
predicative,
diagnostic or prognostic modality.
Another objective of the present invention is to provide methods for
screening for and identifying agents that impact organelles and other
subcellular
compartments in specific ways. When such agents are specific for undesirable
abnormal cells, or for the cells of an undesirable parasites, they are
expected to have
remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic
or disease-
impeditive effects on patients comprising such undesirable cells.
The present invention fulfils these needs and realizes these and other
objectives. Other advantages of the invention are apparent from the
disclosure.
SUMMARY OF THE INVENTION
The present invention is directed in part to methods and compositions for
monitoring cellular processes, conditions and molecules using energy transfer
(ET)
techniques. Such ET-based methods and compositions further provide means to
screen
for and identify agents that alter (e.g., increase or decrease in a
statistically significant
manner) such processes, conditions and molecules. Accordingly, in one aspect
the
invention provides a method for assaying mitochondria) 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
mitochondria) outer membrane, mitochondria) inner membrane, mitochondria)
intermembrane space or mitochondria) 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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
4
concentration of at least one of the energy transfer molecules in the
mitochondria
changes as a function of membrane potential.
In certain embodiments of this aspect of the invention the excited energy
donor molecule transfers energy to the energy acceptor molecule to produce an
excited
energy acceptor molecule, and the signal detected results from energy released
by the
excited energy acceptor molecule. In certain embodiments energy transfer from
the first
energy transfer molecule to the second energy transfer molecule results in a
decrease in
the detectable signal. In certain further embodiments the method comprises
contacting
the mitochondria with an agent that induces dissipation of mitochondria)
membrane
potential. In certain other embodiments the agent that induces dissipation of
mitochondria) membrane potential is an ionophore. In certain further
embodiments the
method comprises contacting the mitochondria with an agent that induces
collapse of
mitochondria) membrane potential. In another embodiment the agent that induces
collapse of mitochondria) membrane potential is CCCP or FCCP. In certain
embodiments the sample is washed prior to the step of detecting a signal, and
in other
embodiments the signal detected is compared with a reference signal. In
certain further
embodiments the reference signal is generated by an indicator of cell number,
an
indicator of mitochondria) mass, an indicator of cellular protein, an
indicator of cellular
DNA, an indicator of mitochondria) DNA, an indicator of mitochondria) protein
and an
indicator of fluid volume.
In other embodiments of the invention, the sample comprises one or
more mitochondria that are present within at least one cell, and the signal
detected is
compared with a reference signal. In certain further embodiments the reference
signal is
generated from a subcellular site that may be a mitochondria) outer membrane,
mitochondria) inner membrane, mitochondria) intermembrane space, mitochondria)
matrix, cytoplasm, nucleus, nuclear membrane or plasma membrane. In another
embodiment the reference signal is generated from extracellular medium. In
another
embodiment mitochondria are present within at least one cell during at least
one step,
and in certain further embodiments the cell is an organism, a cultured cell, a
cybrid cell,
a plant cell or an animal cell. In certain other embodiments the cell is
present in a
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
biological sample derived from a multicellular organism, which in some
embodiments
is a plant cell and in other embodiments is an animal cell; in some
embodiments the
animal is a mammal that in some embodiments is a human. In a further
embodiment
the human has, is suspected of having or is at risk of having a disease or
disorder
5 associated with organellar dysfunction, which in certain further embodiments
is
mitochondria) dysfunction and in certain other embodiments is lysosomal
dysfunction.
In another embodiment of this aspect of the invention, the first energy
transfer molecule localizes to a submitochondrial site that is mitochondria)
matrix or
mitochondria) inner membrane, and the second energy transfer molecule
localizes to a
submitochondrial site that is mitochondria) matrix or mitochondria) inner
membrane. In
one embodiment the concentration of the first energy transfer molecule in the
submitochondrial site does not change as a function of membrane potential, and
the
concentration of the second energy transfer molecule in the mitochondria)
matrix
decreases as a function of membrane potential. In another embodiment the first
energy
transfer molecule has an excitation maximum at a wavelength of from about 373
nm to
about 390 nm, and an emission maximum at a wavelength of from about 400 nm to
about 500 nm; and the second energy transfer molecule has an excitation
maximum at a
wavelength of from about 400 nm to about 500 nm. In a further embodiment 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 mitochondria) matrix or
mitochondria) inner membrane; and the second energy transfer molecule is
DASPEI,
DASPMI, 4-Di-1-ASP, 2-Di-1-ASP, DiOC7(3), DiOC6(3), JC-1 or SYTO~ 18 yeast
mitochondria) stain. In another embodiment the first energy transfer molecule
has an
excitation maximum at a wavelength of from about 425 nm to about 440 nm, and
an
emission maximum at a wavelength of from about 450 nm to about 535 nm; and the
second energy transfer molecule has an excitation maximum at a wavelength of
from
about 450 nm to about 530 nm.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
6
In another embodiment the first energy transfer molecule is a fusion
protein, wherein the fusion protein comprises a cyan-shifted Green Fluorescent
Protein
polypeptide having a mutation in at least one of Phe-64, Ser-65, Tyr-66, Asn-
146, Met-
153, Val-163 and Asn-212, and a polypeptide sequence that localizes the fusion
protein
to a submitochondrial site selected from the group consisting of mitochondria)
matrix
and mitochondria) inner membrane; and the second energy transfer molecule is
DASPEI, 2-Di-1-ASP, DiOC6(3), SYTO~ 18 yeast mitochondria) stain, rhodamine
6G,
JC-I, NBD C6-ceramide or NBD C6-sphingomyelin. In another embodiment the first
energy transfer molecule has an excitation maximum at a wavelength of from
about 470
nm to about 500 nm, and an emission maximum at a wavelength of from about 505
nm
to about 565 nm; and the second energy transfer molecule has an excitation
maximum
at a wavelength of from about 505 nm to about 565 nm.
In yet another embodiment, the first energy transfer molecule is
nonylacridine orange, MitoTracker~ Green FM, MitoFluorTM Green or a fusion
protein,
wherein the fusion protein comprises a Green Fluorescent Protein polypeptide
that is a
wildtype Green Fluorescent Protein polypeptide, a red-shifted Green
Fluorescent
Protein polypeptide having a mutation in one or more of Phe-64, Ser-65, Tyr-
66, Gln-
69, Ser-72 and Thr-203 or a yellow-shifted Green Fluorescent Protein
polypeptide
having a mutation in one or more of Phe-64, Ser-65, Tyr-66, Gln-69, Ser-72 and
Thr-
203, and a polypeptide sequence that localizes the fusion protein to a
submitochondrial
site that is mitochondria) matrix or mitochondria) inner membrane; and the
second
energy transfer molecule is rhodamine 123, JC-l, tetrabromorhodamine 123,
rhodamine
6G, TMRM, TMRE, tetramethylrosamine or rhodamine B. In another embodiment, the
first energy transfer molecule has an excitation maximum at a wavelength of
from about
545 to about 560 nm, and an emission maximum at a wavelength of from about 565
to
about 625 nm; and the second energy transfer molecule has an excitation
maximum at a
wavelength of from about 565 to about 625 nm. In an further embodiment the
first
energy transfer molecule is MitoTracker~ Orange CMTMRos; and the second energy
transfer molecule is DiOC2(5). In another embodiment the first energy transfer
molecule has an excitation maximum at a wavelength of from about 495 to about
510
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
7
nm, and an emission maximum at a wavelength of from about 510 to about 570 nm;
and
the second energy transfer molecule has an excitation maximum at a wavelength
of
from about 510 to about 560 nm. In another embodiment the first energy
transfer
molecule is a fusion protein, wherein the fusion protein comprises a
polypeptide
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 mitochondria) matrix and mitochondria) inner
membrane;
and the second energy transfer molecule is JC-1, tetrabromorhodamine 123,
rhodamine
6G, TMRM, TMRE, tetramethylrosamine, rhodamine B and 4-dimethylamino-
tetramethylrosamine.
In another embodiment of this aspect of the invention, a relative amount
of the signal generated by energy transfer is detected. In certain other
embodiments the
signal is detected over a period of time and a rate of change in the signal
level is
determined, and in certain other embodiments the signal is detected over a
period of
time and integrated. In another embodiment membrane potential comprises an
electric
potential, a pH potential, or both. In one embodiment the first and second
energy
transfer molecules localize to within from about 10 angstroms to about 100
angstroms
of each other, and in another embodiment they localize to within from about 10
angstroms to about 50 angstroms of each other and in another embodiment they
localize
to within from about 20 angstroms to about 50 angstroms of each other. In
certain
embodiments the signal is generated by fluorescence resonance energy transfer.
Turning to another aspect, the present invention provides a method for
identifying an agent that alters mitochondria) 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 mitochondria) outer membrane,
CA 02375542 2001-12-13
WO 00/79274 PCTNS00/17380
8
mitochondria) inner membrane, mitochondria) intermembrane space or
mitochondria)
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 signal
generated in the presence of the candidate agent, and therefrom identifying an
agent that
alters mitochondria) membrane potential.
In another aspect the invention provides a method for identifying a
regulator of an agent that alters mitochondria) membrane potential, comprising
the steps
of contacting, in the absence and presence of a candidate regulator, an agent
that alters
mitochondria) 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
mitochondria) outer membrane, mitochondria) inner membrane, mitochondria)
intermembrane space or mitochondria) 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 regulator to the signal generated in the presence of the candidate
regulator,
and therefrom identifying a regulator of an agent that alters mitochondria)
membrane
potential. In one embodiment the regulator is an agonist of the agent that
alters
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
9
mitochondria) potential, and in another embodiment the regulator is an
antagonist of the
agent that alters mitochondria) potential. In another embodiment the agent
that alters
mitochondria) membrane potential is an apoptogen. In another embodiment the
agent
that alters mitochondria) membrane potential is thapsigargin, an ionophore or
an
excitatory amino acid or derivative thereof. In certain further embodiments
the
ionophore is ionomycin or A2318'7. In certain other embodiments the excitatory
amino
acid or derivative thereof is glutamate, NAAG, NMDA, AMPA, APPA or kainate.
Turning now to another aspect, the invention provides a method for
identifying an agent that preferentially alters mitochondria) membrane
potential in
mitochondria from a first biological source without substantially altering
mitochondria)
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
mitochondria) outer membrane, mitochondria) inner membrane, mitochondria)
intermembrane space or mitochondria) matrix, and the first energy transfer
molecule is
an energy donor molecule and the second energy transfer molecule is an energy
acceptor molecule; exciting the energy donor molecule to produce an excited
energy
donor molecule in the presence of each of the first and second samples;
detecting a
signal generated by energy transfer from the first energy transfer molecule to
the second
energy transfer molecule in the presence of each of the first and second
samples,
wherein the concentration of at least one of the energy transfer molecules in
the
mitochondria changes as a function of membrane potential; and comparing the
signal
generated in the presence of each of the first and second samples in the
absence of the
candidate agent to the signal generated in the presence of each of the first
and second
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
samples in the presence of the candidate agent, and therefrom identifying an
agent that
preferentially alters mitochondria) membrane potential
In one embodiment the first and second biological sources are distinct
biological species, and in another embodiment the first biological source is a
mammal
5 suspected of having, diagnosed as having or predisposed to having a disease,
and the
second biological source is a mammal that is not suspected of having and has
not been
diagnosed as having or predisposed to having the disease. In a further
embodiment the
first biological source is a human and the second biological source is a
human. In
another embodiment the disease is Alzheimer's disease, Parkinson's disease or
type II
10 diabetes.
The present invention provides, in another aspect, a method for
identifying an agent that preferentially alters mitochondria) membrane
potential in
mitochondria from a first biological sample without substantially altering
mitochondria)
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 mitochondria) outer membrane,
mitochondria)
inner membrane, mitochondria) intermembrane space or mitochondria) matrix, and
the
first energy transfer molecule is an energy donor molecule and the second
energy
transfer molecule is an energy acceptor molecule; exciting the energy donor
molecule to
produce an excited energy donor molecule in the presence of each of the first
and
second samples; detecting a signal generated by energy transfer from the first
energy
transfer molecule to the second energy transfer molecule in the presence of
each of the
first and second samples, wherein the concentration of at least one of the
energy transfer
molecules in the mitochondria changes as a function of membrane potential; and
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
11
comparing the signal generated in the presence of each of the first and second
samples
in the absence of the candidate agent to the signal generated in the presence
of each of
the first and second samples in the presence of the candidate agent, and
therefrom
identifying an agent that preferentially alters mitochondria) membrane
potential. In one
embodiment the first tissue and the second tissues are derived from the same
subject,
while in another embodiment the first and second tissues are each derived from
a
subject of the same species. In another embodiment the first and second
tissues are
derived from subjects of distinct species.
It is still another aspect of the invention to provide a method of detecting
the fusion of a first mitochondrion and a second mitochondrion, comprising the
steps of
contacting a first sample comprising one or more mitochondria with a first
energy
transfer molecule that is not endogenous to the mitochondria; contacting a
second
sample comprising one or more mitochondria with a second energy transfer
molecule
that is not endogenous to the mitochondria; wherein the first and second
energy transfer
molecules each localize independently of one another to the same
submitochondrial site
or to acceptably adjacent submitochondrial sites that are mitochondria) outer
membrane,
mitochondria) inner membrane, mitochondria) intermembrane space or
mitochondria)
matrix, and the first energy transfer molecule is an energy donor molecule and
the
second energy transfer molecule is an energy acceptor molecule; contacting the
first
sample with the second sample under conditions and for a time sufficient to
permit
mitochondria) fusion; exciting the energy donor molecule to produce an excited
energy
donor molecule; and detecting a signal generated by energy transfer from the
first
energy transfer molecule to the second energy transfer molecule, and therefrom
determining fusion of the first mitochondrion and the second mitochondrion.
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
12
independently of one another to the same submitochondrial site or to
acceptably
adjacent submitochondrial sites that are mitochondria) outer membrane,
mitochondria)
inner membrane, mitochondria) intermembrane space or mitochondria) 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 mitochondria) 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; and comparing the signal detected in the absence of the candidate
agent to the
signal detected in the presence of the candidate agent, and therefrom
identifying an
agent that alters the fusion of the mitochondria. In certain embodiments the
agent
increases mitochondria) membrane potential, in certain other embodiments the
agent
dissipates mitochondria) membrane potential, in certain other embodiments the
agent
collapses mitochondria) membrane potential, and in certain embodiments the
agent
alters an equilibrium distribution of at least one ionic species on either
side of a cellular
membrane. In a further embodiment the ionic species is Ca+Z and the cellular
membrane is a mitochondria) membrane. In certain embodiments the agent that
collapses mitochondria) membrane potential is an apoptogen, and in certain
other
embodiments the agent that collapses mitochondria) membrane potential
interacts with
an adenine nucleotide translocator, and in certain other embodiments the agent
that
collapses mitochondria) membrane potential is atractyloside,
carboxyatractyloside,
bongkrekic acid or isobongkrekic acid.
Turning to another aspect, the invention provides a reagent for
measuring mitochondria) O~r, 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 Dyr and the accumulation of the other of the molecules in
mitochondria is
independent of Dyr. In one embodiment the molecule that accumulates in
mitochondria
independent of Ayr is NAO, MitoTracker~ Green FM, MitoFluorTM, DAPI, or a
fusion
protein comprising a polypeptide that is a red- shifted Green Fluorescent
Protein
CA 02375542 2001-12-13
WO 00/79274 PCTNS00/17380
13
polypeptide, a yellow-shifted Green Fluorescent Protein polypeptide or a
"FLASH"
polypeptide, and a polypeptide sequence that localizes the fusion protein to
the
mitochondria) matrix or inner membrane. In certain other embodiments the
molecule
that accumulates in mitochondria in a manner dependent on OW is TMRM, TMRE,
rhodamine 123, ethidum bromide, 4-Di-1-ASP, 2-Di-1-ASP or DASPEI. The
invention
also provides, in certain embodiments, a kit comprising the reagent just
described and
ancillary reagents for measuring mitochondria) Ayr.
It is another aspect of the present invention to provide a method for
assaying cellular membrane potential, comprising the steps of: contacting a
sample
comprising at least one cellular membrane, simultaneously or sequentially and
in either
order, with each of a first and a second energy transfer molecule that is not
endogenous
to the sample, wherein 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
1 ~ cellular membrane that forms a subcellular compartment, and the first
energy transfer
molecule is an energy donor molecule and the second energy transfer molecule
is an
energy acceptor molecule; exciting the energy donor molecule to produce an
excited
energy donor molecule; and detecting a signal generated by energy transfer
from the
first energy transfer molecule to the second energy transfer molecule, wherein
the
concentration of at least one of the energy transfer molecules in the membrane
site
changes as a function of membrane potential. In one embodiment the first
energy
transfer molecule localizes to a first membrane site that is mitochondria,
endoplasmic
reticulum, Golgi, lysosome or plasma membrane and the second energy transfer
molecule localizes to the same membrane site or to an acceptably adjacent
membrane
site that is mitochondria, endoplasmic reticulum, Golgi, lysosome or plasma
membrane.
In another embodiment the concentration of the first energy transfer molecule
in the
first membrane site does not change as a function of membrane potential, and
the
concentration of the second energy transfer molecule in the membrane site
decreases as
a function of membrane potential.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
14
In one embodiment the first energy transfer molecule has an excitation
maximum at a wavelength of from about 373 nm to about 390 nm, and an emission
maximum at a wavelength of from about 400 nm to about 500 nm; and the second
energy transfer molecule has an excitation maximum at a wavelength of from
about 400
nm to about 500 nm. In a further embodiment the first energy transfer molecule
has an
excitation maximum at a wavelength of from about 425 nm to about 440 nm, and
an
emission maximum at a wavelength of from about 450 nm to about 535 nm; and the
second energy transfer molecule has an excitation maximum at a wavelength of
from
about 450 nm to about 530 nm. In another embodiment the first energy transfer
molecule has an excitation maximum at a wavelength of from about 470 nm to
about
500 nm, and an emission maximum at a wavelength of from about 505 nm to about
565
nm; and the second energy transfer molecule has an excitation maximum at a
wavelength of from about 505 nm to about 565 nm. In another embodiment the
first
energy transfer molecule has an excitation maximum at a wavelength of from
about 545
to about 560 nm, and an emission maximum at a wavelength of from about 565 to
about
625 nm; and the second energy transfer molecule has an excitation maximum at a
wavelength of from about 565 to about 625 nm.
In yet another aspect, the invention provides a method for identifying an
agent that alters a cellular membrane potential, comprising the steps of
contacting, in
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
one of the energy transfer molecules in the subcellular compartment changes as
a
function of membrane potential; and comparing the signal generated in the
absence of
the candidate agent to the signal generated in the presence of the candidate
agent, and
therefrom identifying an agent that alters cellular membrane potential.
5 Another aspect of the invention is to provide a method for identifying a
regulator of an agent that alters cellular membrane potential, comprising the
steps of
contacting, in the absence and presence of a candidate regulator, an agent
that alters a
cellular membrane potential (which may be an agent identified according to the
method
just described) and a sample comprising one or more cellular membranes
10 simultaneously or sequentially and in either order with each of a first and
a second
energy transfer molecule that is not endogenous to the sample, wherein the
first and
second energy transfer molecules each localize independently of one another to
the
same membrane site or to acceptably adjacent membrane sites such that at least
one of
the energy transfer molecules localizes to a cellular membrane that forms a
subcellular
15 compartment, and the first energy transfer molecule is an energy donor
molecule and
the second energy transfer molecule is an energy acceptor molecule; exciting
the energy
donor molecule to produce an excited energy donor molecule; detecting a signal
generated by energy transfer from the first energy transfer molecule to the
second
energy transfer molecule, wherein the concentration of at least one of the
energy
transfer molecules in the subcellular compartment changes as a function of
membrane
potential; and comparing the signal generated in the absence of the candidate
regulator
to the signal generated in the presence of the candidate regulator, and
therefrom
identifying a regulator of an agent that alters cellular membrane potential.
In another aspect the invention provides a method for identifying an
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
16
endogenous to the sample, wherein the first sample is derived from a first
biological
source and the second sample is derived from a second biological source that
is distinct
from the first biological source, the first and second energy transfer
molecules each
localize independently of one another to the same membrane site or to
acceptably
adjacent membrane sites such that at least one of the energy transfer
molecules localizes
to a cellular membrane that forms a subcellular compartment, and the first
energy
transfer molecule is an energy donor molecule and the second energy transfer
molecule
is an energy acceptor molecule; exciting the energy donor molecule to produce
an
excited energy donor molecule in the presence of each of the first and second
samples;
detecting a signal generated by energy transfer from the first energy transfer
molecule to
the second energy transfer molecule in the presence of each of the first and
second
samples, wherein the concentration of at least one of the energy transfer
molecules in
the subcellular compartment changes as a function of membrane potential; and
comparing the signal generated in the presence of each of the first and second
samples
in the absence of the candidate agent to the signal generated in the presence
of each of
the first and second samples in the presence of the candidate agent, and
therefrom
identifying an agent that preferentially alters cellular membrane potential
Turning to another aspect, the invention provides a method for
identifying an agent that preferentially alters a cellular membrane potential
in a
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
17
molecule is an energy donor molecule and the second energy transfer molecule
is an
energy acceptor molecule; exciting the energy donor molecule to produce an
excited
energy donor molecule in the presence of each of the first and second samples;
detecting a signal generated by energy transfer from the first energy transfer
molecule to
the second energy transfer molecule in the presence of each of the first and
second
samples, wherein the concentration of at least one of the energy transfer
molecules in
the subcellular compartment changes as a function of membrane potential; and
comparing the signal generated in the presence of each of the first and second
samples
in the absence of the candidate agent to the signal generated in the presence
of each of
the first and second samples in the presence of the candidate agent, and
therefrom
identifying an agent that preferentially alters a cellular membrane potential.
In still another aspect the invention provides a method for detecting a
specific type of cell in a sample, comprising the steps of contacting a sample
comprising one or more mitochondria simultaneously or sequentially and in
either order
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. In one embodiment 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. In
another
embodiment the specific type of cell is a cancer cell.
In another aspect the invention provides a method for identifying a Dym
stabilizing agent, comprising the steps of contacting, in the absence and
presence of a
candidate Dyrm stabilizing agent, an agent that alters ~yrm and a sample
comprising one
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
18
or more mitochondria simultaneously or sequentially and in either order with
each of a
first and a second energy transfer molecule that is not endogenous to the
mitochondria,
wherein the first and second energy transfer molecules each localize
independently of
one another to the same submitochondrial site or to acceptably adjacent
submitochondrial sites that are mitochondria) outer membrane, mitochondria)
inner
membrane, mitochondria) intermembrane space or mitochondria) 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 ~yrm stabilizing agent, to the
signal generated
in the presence of the candidate ~yJm stabilizing agent, and therefrom
identifying ~yJm
stabilizing agent. In one embodiment the mitochondria are contained within
cells, and
in a further embodiment the agent that alters ~Wm 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 Ayr", stabilizing agent identified according to the
method just
described. In another embodiment, the invention provides a method of treating
stroke
comprising administering the Dyrm stabilizing agent to a patient in need
thereof.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description and attached drawings.
All
references disclosed herein are hereby incorporated by reference in their
entirety as if
each was incorporated individually.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
19
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 schematically depicts direct and indirect methods for measuring
energy transfer. Symbols: "~,EX," peak excitation wavelength; "7~EM," peak
emission
wavelength; "e," energy; open box, receptive filter setting; closed box,
closed filter
setting.
Figure 2 schematically depicts submitochondrial structural
compartments and energy transfer interactions between energy transfer donor
and
acceptor molecules in designated compartments: "CS," cytosolic space; "OM,"
outer
membrane; "IS," intermembrane space; "IM," inner membrane; "MX," matrix;
"DMx,"
donor compound localizing to the matrix; "AIM," acceptor compound localizing
to the
inner membrane; "DIS," donor compound localizing to the intermembrane space;
"e,"
energy.
Figure 3 shows representative data from FRET-based assays of ~yf",.
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 Dyrm.
Figure 5 shows calibration of the concentrations of an ET donor
molecule (NAO) and an ET acceptor molecule (TMRM) in FRET assays of ~yfm.
Figure 6 shows time-course data from a FRET assay of Dye", using NAO
and TMRM alone and in combination.
Figure 7 shows Type I FRET ~W", 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 Dym assay of various agents. Symbols:
"MO," media (HBSS) only; "C," CCCP; "I+RR," ionomycin and ruthenium red.
Figure 9 shows Type I FRET Ayr", 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 Dy collapsing agent CCCP.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
Figure 11 shows Type II FRET Dym assay. Symbols: "MO," results
from samples treated with media (HBSS) only; "4BA;" results from samples
treated
with the ~Wm-dissipating agent 4-bromo-A23187; "C," and arrow indicate time of
CCCP addition to samples.
5 Figure 12 is a dose response curve for a Dyr-dissipating compound
(ionomycin).
Figure 13 is a dose response curve for a compound (cyclosporin A) that
protects mitochondria against a Dy-dissipating compound (ionomycin).
Figure 14 shows a dose-response curve of three cell lines to the ~yrm-
10 dissipating agent A-23187.
Figure 15 shows FRET in carcinoma cells following experimentally
induced loss of mitochondria) membrane potential.
Figure 16 shows the concentration-dependent response of permeabilized
cells to calcium ions, which leads to a collapse of OW at higher
concentrations of Ca'+.
15 Figure 17 shows the same data presented in Figure 16 wherein mean
values are plotted without error bars. Also, the response of permeabilized
cells to
CCCP, an agent known to induce Dye collapse, is not shown in Figure 16 but is
presented here. It is noteworthy that, at a concentration of 100 uM, Ca2+
induces
collapse of Dyr in a manner that is roughly equivalent, in terms of both the
extent of
20 response and time course, to that seen in cells treated with CCCP.
Figure 18 is a concentration response curve (CRC) of Ca2+ in
permeabilized cells that was generated from the data presented in Figures 16
and 17.
Figure 19 is a CRC of RU-360, an inhibitor of the mitochondria) calcium
uniporter, in permeabilized cells that were also contacted with Ca2+.
Figure 20 is a CRC of cyclosporin A, an agent known to modulate Caz+-
induced O~r collapse, in permeabilized cells that were also contacted with
Ca2+.
Figure 21 shows a CRC of oligomycin, a specific ATP synthase
inhibitor, in permeabilized SH-SYSY cells.
Figure 22 shows a CRC of ADP in permeabilized SH-SYSY cells.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
21
Figure 23 shows a CRC of bongkrekic acid in permeabilized SH-SYSY
cells.
Figure 24 shows a CRC of nigericin in permeabilized SH-SYSY cells.
SYMBOLS AND ABBREVIATIONS
Descriptions of specialized terms and abbreviations are listed in Table 1.
Unless otherwise, indicated, symbols for nucleotides and amino acids are as
described
in 37 ~ C.F.R. 1.821.
Term or If Chemical or Instrument:
AbbreviationDescription or Formula
(if any)
ame of Supplier(s)'~
4~I', 0'>'mmitochondria) membrane potential---
OpH pH potential ---
A-23187 1-(4,5-dimethoxy-2-nitrophenyl)ethylCalbiochem
ester
4-BA 4-bromo A-23187 Calbiochem
ANT adenine nucleotide translocator---
AO acridine orange MP
ATR atractyloside Sigma
BKA bongkrekic acid Biomol, Calbiochem
--- BODIPY~ TR ceramide MP
--- BODIPY~ FL Br2 C5-ceramide MP
--- BODIPY~ FL CS-ceramide MP
--- BODIPY~ FL CS- sphingomyelin MP
--- BODIPY~ FL conjugate isomer MP
1
BFA brefeldin A from Penicillium MP
brefeldianum
calcein (a.k.a. fluorexon, fluoresceinMP, Sigma
complexon)
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
22
Term or If Chemical or Instrument:
AbbreviationDescription or Formula
Name of Supplier(s)X
(if any)
CATR carboxyatractyloside Calbiochem
CO-Fluro 5-carboxyfluorescein MP
CCCP carbonyl cyanide m-chlorophenyl-Sigma
hydrazone
CsA cyclosporin A Calbiochem
DAPI 4',6-diamidino-2-phenylindoleMP
DASPEI 2-(4-(dimethylamino)styryl)-N-MP
ethylpyridinium iodide
DASPMI dimethylaminostyrylmethylpyridiniumMP
iodide; comprises 2 isomers,
2-Di-I-
ASP and 4-Di-1-ASP
2-Di-1-ASP 2-(4-(dimethylamino)styryl)-N-MP
methylpyridinium iodide
4-Di-I-ASP 4-(4-(dimethylamino)styryl)-N-MP
methylpyridinium iodide
DilC~6(3) I,I'-dihexadecyl-3,3,3',3'-tetramethyl-MP
indocarbocyanine perchlorate
DiIC~B(3) 1,I'-dioctadecyl-3,3,3',3'-tetramethyl-MP
indocarbocyanine perchlorate
--- 4-dimethylamino-tetramethylrosamineMP
DiOC2(5) 3,3'-diethyloxadicarbocyanineMP
iodide
DiOCS(3) 3,3'-dipentyloxacarbocyanine MP
iodide
DiOC6(3) 3,3'-dihexyloxadicarbocyanineMP
iodide
DiOC~(3) 3,3'-diheptyloxadicarbocyanineMP
iodide
EtBr ethidium bromide Sigma
ET energy transfer - - -
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
23
Term or If Chemical or Instrument:
AbbreviationDescription or Formula
Name of Supplier(s)X
(if any)
ETC electron transport chain ---
FCCP carbonyl cyanide p- S i g m a
(trifluoromethoxy)phenyl-hydrazone
FLASH fluorescein arsenical helix ---
binder
FLIPRTM Fluorometric Imaging Plate Mol. Dev.
Reader
FRET fluorescence resonance energy---
transfer
FL1N-1TM (proprietary compound) MP
--- hydroxystilbamidine, methanesulfonateMP
JC-1 5,5',6,6'-tetrachloro-1,1',3,3'-tetra-MP
ethylbenzimidazoylcarbocyanine
iodide
lucigenin bis-N-methylacridinium nitrateMP
LysoSensorTM(proprietary compounds) MP
s
LysoTracker (proprietary compounds) MP
TMS
MELAS Mitochondria) Encephalopthy,---
Lactic
Acidosis and Stroke
MixCon Mixed Controls (cybrids) ---
Mpp+ 1-methyl-4-phenylpyridinium Calbiochem, RBI
MPT Mitochondria) Permeability ---
Transition
mtDNA mitochondria) DNA ---
MitoFluorTMs(proprietary compounds) MP
MitoTracker (proprietary compounds) MP
~s
NAO 10-N nonyl acridine orange MP
--- NBD C6-ceramide MP
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
24
Term or If Chemical or Instrument:
~~bbreviationDescription or Formula
(if any) Name of Supplier(s)X
--- NBD C6-sphingomyelin MP
--- oligomycin C a 1 b i o c h a m
PI propidium iodide S i g m a
PMF protonmotive force ---
rh123 rhodamine 123 MP, Calbiochem
rhB rhodamine B MP
rh6G rhodamine 6G MP
RR ruthenium red (ammoniated Sigma
ruthenium
oxychloride)
SNAFL~ seminapthofluorescein calceinMP
calcein
SYTO~ 18 (proprietary compound) MP
TB-rh123 tetrabromorhodamine 123 MP
TMRE tetramethylrhodamine, ethyl MP
ester
TMRM tetramethylrhodamine, methyl MP
ester
--- tetramethylrosamine MP
--- 4-dimethylaminotetramethylrosamineMP
--- thapsigargin Calbiochem
--- valinomycin Calbiochem
Green Fluorescent vectors from Aurora
Proteins /
Clontech
GFP green fluorescent protein ---
BFP blue-shifted green fluorescent---
protein
CFP cyan-shifted green fluorescent---
protein
RFP red-shifted green fluorescent---
protein
YFP yellow-shifted green fluorescent---
protein
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
* Abbreviations for suppliers: "Calbiochem", Calbiochem, Inc., La Jolla, CA;
"MP,"
Molecular Probes, Inc., Eugene, OR; "Biomol," Biomol Research :Laboratories,
Inc.,
Plymouth Meeting, MA; "Mol. Dev.," Molecular Devices, Sunnyvale, CA; "Aurora,"
Aurora Biosciences Corp., San Diego, CA; "Clontech," CLONTECH Laboratories,
Inc.,
5 Palo Alto, CA; "Sigma," Sigma Chemical Co., St. Louis, MO; RBI, Research
Biochemicals International, Natick, MA.
DETAILED DESCRIPTION OF THE INVENTION
The present invention pertains in part to the use of intermolecular energy
transfer to monitor intracellular and intraorganellar conditions. In
particular, the
10 invention derives from the unexpected observation that such intracellular
and
intraorganellar conditions can be surveyed using energy transfer molecule
donor-
acceptor pairs that need not undergo specific intermolecular recognition
events such as
affinity binding interactions. Rather, according to the present disclosure,
under
particular naturally occurring or artificially induced intracellular and/or
intraorganellar
15 physiologic conditions, appropriately paired energy transfer donor and
acceptor
molecules can be selected that accumulate at acceptably adjacent sites as
provided
herein, to generate detectable signals.
By way of background, energy transfer (ET) is generated from a
resonance interaction between two molecules: an energy-contributing "donor"
molecule
20 and an energy-receiving "acceptor" molecule. Energy transfer can occur when
(1) the
emission spectrum of the donor overlaps the absorption spectrum of the
acceptor and
(2) the donor and the acceptor are within a certain distance (for example,
less than about
10 nm) of one another. The efficiency of energy transfer is dictated largely
by the
proximity of the donor and acceptor, and decreases as a power of 6 with
distance.
25 Measurements of ET thus strongly reflect the proximity of the acceptor and
donor
compounds, and changes in ET sensitively reflect changes in the proximity of
the
compounds such as, for example., association or dissociation of the donor and
acceptor.
According to the present invention, both energy transfer molecules, the
ET donor molecule and the ET acceptor molecule, are molecules that are not
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
26
endogenous to the sample as provided herein (by way of non-limiting example, a
cell,
an organelle such as a mitochondrion, or a subcellular or suborganellar
compartment)
with which they are contacted. The donor and acceptor compounds may co-
localize to a
subcellular compartment in such a manner as to achieve sufficient proximity to
one
another for a particular type of energy transfer to occur. In certain aspects
of the
invention, such co-localization may be dependent upon, or may be disrupted by,
intracellular processes or responses to chemical agents. For instance, such
processes or
responses can lead to, respectively, an increase or a decrease in energy
transfer that can
be detected, for example, by detecting a signal. Thus, for example, detection
of the
degree or rate of energy transfer between the ET donor and ET acceptor
molecules may
provide in pertinent part a method for assaying a given intracellular process
or response.
In certain preferred embodiments the invention provides a method for assaying
a
cellular membrane potential, and in certain other preferred embodiments the
invention
provides a method for assaying mitochondrial membrane potential.
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. Optionally, the sample may be washed
under
suitable conditions prior to the step of detecting a signal, for example to
improve
sensitivity for detecting the signal. Those having ordinary skill in the art
can readily
determine the manner by which the sample is contacted, in view of the
properties of the
sample and of the ET molecules selected, and in view of the teachings provided
herein.
As also provided herein, the subject invention method can employ any suitable
ET
donor molecule and ET acceptor molecule that can function as a donor-acceptor
pair.
As discussed in greater detail below, the method of the present invention may
be used to
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
27
identify an agent that alters a cellular membrane potential, or to identify a
molecule that
is a regulator of such an agent.
In certain preferred embodiments the invention is directed to a method
for assaying mitochondrial membrane potential, wherein neither the ET donor
molecule
nor the ET acceptor molecule is endogenous to mitochondria, and wherein the ET
donor
and the ET acceptor each localize independently of one another to the same
submitochondrial site or to acceptably adjacent submitochondrial sites as
provided
herein.
Optionally, in preferred embodiments the ET donor molecule and the ET
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
absorption of radiation at one wavelength ("excitation"), followed by nearly
immediate
re-radiation ("emission"), usually at a different wavelength, that ceases
almost at once
when the incident radiation stops. At a molecular level, fluorescence occurs
as certain
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, and "bioluminescence" refers
to the
emission of light from-- living organisms or cells, organelles or extracts
derived
therefrom.
In certain preferred embodiments, a detectable signal that is generated by
energy transfer between ET donor and acceptor molecules results from
fluorescence
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
28
resonance energy transfer (FRET). FRET occurs within a molecule, or between
two
different types of molecules, when energy from an excited donor fluorophore is
transferred directly to an acceptor fluorophore (for a review, see Wu et al.,
Analytical
Biochem. 218:1-13, 1994). In general, the energy transfer from an excited
fluorophore
(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). 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 ).
The use of 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, is known in the art. When measuring such intermolecular
interactions, the ET donor and acceptor fluorophores are typically situated on
two
different molecules that are known or believed to enter into close association
with each
other. On the other hand, when intramolecular interactions are measured, the
ET donor
and acceptor fluorophores are present on the same molecule.
In contrast to such known uses of FRET methodologies, wherein ET
donor and acceptor fluorophores are brought into proximity with each other
through
known specific molecular interactions, the present invention is based on the
unexpected
observation that energy transfer can occur between ET donor and ET acceptor
CA 02375542 2001-12-13
WO 00/79274 PCTNS00/17380
29
fluorophores that are brought into proximity with one another by virtue of
their having
selectively concentrated or accumulated in a common subcellular compartment,
for
example, an organelle, a sub organellar site or other subcellular locale. As a
result, the
present invention can be used to monitor a variety of conditions or processes
within, or
associated with, such subcellular compartments.
As provided herein, contemplated uses of the invention include but need
not be limited to (i) monitoring conditions and processes within subcellular
compartments, (ii) monitoring interactions between pairs of macromolecules
found
within or associated with such subcellular compartments, (iii) identifying
agents that
influence subcellular compartments and/or intracellular processes in a species-
specific
manner, and (iv) identifying agents that influence subcellular compartments
and/or
intracellular processes in such a manner as to treat diseases and disorders of
mammals
and other animals, including humans, and plants. Each of these uses is
described in
greater detail below.
Typically, the invention relates in part to a method for assaying a
sample, which in preferred embodiments is a biological sample and in
particularly
preferred embodiments is a biological sample containing one or more
mitochondria. In
other preferred embodiments the biological sample contains one or more
cellular
membranes, including the plasma membrane and intracellular membrane bounded
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.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
In certain embodiments of the invention, it may be preferred to use intact
cells whereas, in certain other embodiments, the use of permeabilized cells
may be
preferred. A permeabilized cell is a cell that has been treated in a manner
that results in
a partial or complete loss of plasma membrane selective permeability. As a
first
5 example, it may be desirable to permeabilize a cell in a manner that permits
calcium
cations in the extracellular milieu to diffuse into permeabilized cells and
contact
mitochondria. Thus, in this instance, permeabilization serves as an
alternative to the use
of a calcium ionophore. As a second example, certain detectably labeled
molecules,
such as certain of the ET donor and/or ET acceptor molecules provided herein,
may
10 penetrate the plasma membrane at a moderate rate, or to a limited degree,
unless their
entry into the cytosol is facilitated in some manner. Permeabilization of
cells is one
manner by which the cytosolic entry of such ET molecules can be facilitated.
As a third
example, some candidate agents being tested according to the method may
penetrate the
plasma membrane at a moderate rate, or to a limited degree, unless their entry
into the
15 cytosol is facilitated in some manner. Permeabilization of cells is one
manner by which
the entry of such candidate agents into the cytosolic space can be
facilitated. Active
agents that are identified under these conditions can subsequently be modified
chemically to enhance their uptake by whole cells; active agents that are so
modified are
expected to serve as lead compounds for drug development and, in some
instances, may
20 themselves be used as drugs or as drug candidates.
Those having ordinary skill in the art are familiar with methods for
permeabilizing cells, for example by way of illustration and not limitation,
through the
use of surfactants, detergents, phospholipids, phospholipid binding proteins,
enzymes,
viral membrane fusion proteins and the like; by exposure to certain bacterial
toxins,
25 such as a-hemolysin; by contact with hemolysins such as saponin (which is
also a
nonionic detergent, as is digitonin); through the use of osmotically active
agents; by
using chemical crosslinking agents; by physicochemical methods including
electroporation and the like, or by other permeabilizing methodologies
including, e.g.,
physical manipulations such as electroporation. Those skilled in the art are
familiar
30 with methods for permeabilizing cells and can readily determine without
undue
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
31
experimentation the most appropriate permeabilizing agent for use according to
the
present invention as provided herein. Relevant factors for this determination
include
but are not limited to toxicity of the permeabilizing agent to a specific
cell, the
molecular size of the molecule for which entry into the cell is sought through
the use of
permeabilization, and the like (see, e.g., Schulz, Methods Enzymol. 192:280-
300, 1990).
Thus, for instance, cells may be permeabilized using any of a variety of
known techniques, including addition of permeabilizing agents such as
bacterial toxins,
for example, streptolysin O, Staphylococcus aureus a-toxin (a.k.a. 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 (3-D-glucoside and the like) at
concentrations
below those used to lyse cells and solubilize membranes (i. e., below the
critical micelle
concentration). Certain common transfection reagents, such as DOTAP, may also
be
used. ATP can also be used to permeabilize intact cells, as may be low
concentrations
of chemicals commonly used as fixatives (e.g., formaldehyde). All of the
permeabilizing agents described in this paragraph are available from, e.g.,
Sigma
Chemical Co., St. Louis, MO (see Sigma catalog entitled "Biochemicals and
Reagents
for Life Science Research," Anon., 1999, and references cited therein for
these and other
permeabilizing agents).
In certain embodiments of the invention, the subject or biological source
may be suspected of having or being at risk for having a disease associated
with
organellar dysfunction including altered mitochondria) function and
mitochondria)
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.
Examples of
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
32
organellar dysfunction may include, but need not be limited to, lysosomal
storage
defects such as the mucopolysaccaridoses, I-cell disease, Wolman disease and
cholesteryl ester storage disease (e.g., Du et al., 1998 Mol. Genet. Metab.
64:126-34);
plasma membrane defects such as ion channel dysfunction in cystic fibrosis;
endoplasmic reticulum storage diseases (e.g., Kim and Arvan, 1998 Endocr. Rev.
19:173-202); diseases associated with Golgi defects (e.g., ALS, AD, Gonatas et
al.,
1998 Histochem. Cell. Biol. 109:591-600) and other types of organellar
dysfunction that
are known to those familiar with the art.
In certain preferred embodiments it may be desirable to compare the
signal detected according to the method of the invention with a reference
signal.
Selection of a suitable reference signal will according to criteria with which
those
having ordinary skill in the art will be familiar, and may vary depending on
the
particular cellular membrane being assayed and upon the particular donor-
acceptor pair
employed. For example, a reference signal may be generated by a reference
compound
such as an ET donor or ET acceptor molecule or a distinct reporter molecule
that is an
indicator as provided herein, and may further be generated in the absence or
presence of
a sample. Such reporter molecules or indicators may include a detectable
compound
that can be detected as indicative of one or more of a quantity of a
detectable
component or a location of a detectable component, or the like. For example,
by way of
illustration and not limitation, a reference signal may be generated by a
reporter
molecule that permits normalization of a detected energy transfer signal
according to
the number of cells present (e.g., the reporter may be any of numerous known
indicators
of cell number, such as selective stains for cell nuclei, for example,
propidium iodide or
ethidium bromide).
In certain other embodiments, the reference signal is generated by an
indicator of the mitochondria) mass, the mitochondria) number or the
mitochondria)
volume present. For example, where an indicator of mitochondria) mass is
selected, a
reporter molecule such as nonylacridine orange (which can also be an ET donor)
may
be employed. Methods for quantifying mitochondria) mass, volume and/or
mitochondria) number are known in the art, and may include, for example,
quantitative
CA 02375542 2001-12-13
WO 00/79274 PCT/LJS00/17380
33
staining of a representative biological sample. Typically, quantitative
staining of
mitochondria) may be performed using organelle-selective probes or dyes,
including but
not limited to mitochondrion selective reagents such as fluorescent dyes that
bind to
mitochondria) molecular components (e.g., nonylacridine orange,
MitoTrackersT~''') or
potentiometric dyes that accumulate in mitochondria as a function of
mitochondria)
inner membrane electrochemical potential (see, e.g., Haugland, 1996 Handbook
of
Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes,
Eugene,
OR). As another example, mitochondria) 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). These or any other
means
known in the art for quantifying mitochondria) mass, volume and/or
mitochondria)
number in a sample are within the contemplated scope of the invention. For
example,
the use of such quantitative determinations for purposes of calculating
mitochondria)
density is contemplated and is not intended to be limiting. In certain highly
preferred
embodiments, mitochondria) protein mass in a sample is determined using well
known
procedures. For example, a person having ordinary skill in the art can readily
prepare
an isolated mitochondria) 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.
In other embodiments, a reference signal may be generated by a reporter
molecule that permits normalization of a detected energy transfer signal
according to
the amount of protein present (e.g., coomassie blue, fluorescamine,
bicinchoninic acid)
or to the amount of nucleic acid present (e.g., ethidium bromide, acridine
orange,
methylene blue). As another example, a reference signal may be generated by a
detectable reporter molecule that is soluble in a liquid medium containing the
sample,
but that cannot traverse cellular membranes and so serves as a marker of
extracellular
medium, for example as an indicator of fluid volume. For example, where
extraordinarily sensitive instrumentation (e.g., see infra) may be used to
detect ET
signals, such an indicator may permit improved quantitative precision by
calibration/
normalization of sample volumes. Many compounds that are suitable for use as
such
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
34
reference signals will be known to those familiar with the art, who may select
such
compounds as sources of a reference signal in a manner dependent on, inter
alia, the
particular cellular membrane potential being assayed and the particular donor-
acceptor
pair employed.
As used herein, detecting a "relative amount" of a signal may include but
is not limited to detecting a signal for purposes of comparing it to a
reference signal as
provided above. Thus, detecting a relative amount of a signal may refer to
detecting
only a portion of a signal (e.g., detecting a signal at less than 100%
efficiency), or to
detecting a signal only a portion of which is generated by energy transfer, or
to
detecting a portion of a signal relative to a signal detected from another
sample such as
a control sample, regardless of whether any of such other signals detected are
reference
signals as provided herein. Detection of a signal according to the methods
disclosed
herein may include quantification of ET by conventional or arbitrarily
assigned units of
measure. In certain embodiments, a signal may be detected over a period of
time such
that one or more behaviors of the signal may be analyzed as a function of
time. For
instance, in some embodiments described herein, a signal may be detected over
a period
of time, which refers to any method of detecting a sample in a manner that
provides
more than a single detection event, such that a correlation of a detected
signal with a
discrete point in time can be established. Thus, for example, in certain
embodiments a
change in an amount of a signal may be detected over two or more time points,
and a
rate of change in the level of signal is determined (e.g., a slope or a rate-
of change of a
slope such as a first order derivative is determined, when the signal level is
plotted as a
function of time). As another example, in certain other embodiments an amount
of a
signal may be cumulatively determined over a discrete time interval, to
provide a
summed signal (e.g., an integrated signal). These and other techniques known
in the art
for analyzing quantitative data, and in particular for analyzing such data
having a
temporal component, are within the contemplated invention and are described in
greater
detail below.
Thus, any of the methods provided by the invention can be modified so
as to also include a reference signal that correlates with a reference
parameter of interest
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
for the purpose of, e.g., standardizing for cell number, quantity of cellular
protein or
cellular nucleic acids, mitochondria) mass, quantity of mitochondria) protein
or
mitochondria) nucleic acids, indicator of fluid volume or the like. The
reference signal,
which can be used as an internal standard, need not result from energy
transfer and can
5 involve any signal that can be correlated with the desired reference
parameter but which
does not interfere with detection of the test/assay signal. In the context of
the invention,
a reference compound can interfere with the test/assay signal if it generates
a signal that
cannot be resolved from the test/assay signal, or if it localizes to the same
subcellular
compartment as the ET donor and acceptor compounds and itself acts as an ET
acceptor
10 or donor compound.
An instrument such as FLIPRTM can be set to alternate between reading
signals at two different wavelengths with a cycling time of about one second;
in this
manner, the reference signal and the test/assay signal (e.g., FRET, 4y~) can
be read over
the same time course. However, the reference need not be read at the same time
as the
15 test/assay signal. For example, in some aspects of the invention, it is
necessary to
disrupt the cells in order to detect the reference signal. and this typically
necessitates
that the reference signal be read after the test or assay has been completed.
Some non-limiting examples of reference signals include the following.
After the test or assay, as is known in the art, cellular protein (including
mitochondria)
20 protein) can be measured using methods such as the Bradford or Lowry
assays, and
nucleic acid can be measured via the use of fluorescent dyes such as propidium
iodide
(PI). Nucleic acids can also be measured in living cells. For example, in
digitonin-
permeabilized cells, propidium iodide (PI; peak excitation, 536 nm; peak
emission, 617
nm when bound to a nucleic acid) binds nuclear and cytoplasmic nucleic acids
but
25 cannot access the mitochondria) matrix and the mitochondria) nucleic acids
contained
therein; PI thus provides a reference signal for quantity of cellular nucleic
acids. The
permeant compound acridine orange (AO) can be used in living cells to
distinguish
RNA and DNA as it has distinct excitation/emission spectra depending on the
type of
nucleic acid to which it is bound (AO:DNA, peak excitation, 500 nm; peak
emission,
30 526 nm; AO:RNA, peak excitation, 460 nm; peak emission, 650 nm). The SYTO
stains
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
36
can also be used to detect nucleic acids in living cells; the manufacturer
(Molecular
Probes, Inc., Eugene, OR) of the SYTO stains indicates that all of the SYTO
stains can
access nuclear and cellular nucleic acids and some can also access
mitochondria)
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 mitochondria) mass in living cells (Mancini et al., Ann. Surg.
Oncol. 5:287-
295, 1998; Vayssiere et al., In Vitro Cell. Dev. Biol. 28A:763-772, 1992,
respectively).
The present invention provides diagnostic and prognostic methods, as
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 mitochondria) membrane potential. Diagnostic uses include methods for
assaying a cellular process or condition (e.g., a cellular membrane potential
such as
mitochondria) 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. Prognostic uses include
methods
wherein a biological sample comprising a cellular membrane or subcellular
compartment is taken from a patient known to have a disease or disorder in
which the
monitored intracellular process or condition is altered. In such prognostic
uses, for
example, biological samples from the patient are prepared and tested for their
response
to agents known to impact the monitored intracellular process or condition in
some, but
not all, instances. A desired response of the biological sample to a
particular agent
indicates that the patient from which the sample was taken will respond best
to a
treatment that correlates with positive response to that treatment. In a
related aspect,
pharmacogenetic studies using the invention are employed to determine the
correlations
between different treatments and specific measurements generated by the
invention.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
37
Non-limiting examples of diseases or disorders that are thought to
involve the altered function or dysfunction of subcellular compartments
include
Alzheimer's disease, Parkinson's disease, type II diabetes and lysosomal
storage
disorders. When the subcellular compartment of interest is the mitochondrion,
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 2-16:500-503, 1989), Chomyn et al.
(Mol.
Cell. Biol. 11:2236-2244, 1991 ), Miller et al. (J. Neurochem. 67:1897-1907,
1996),
Swerdlow et al. (Annals of Neurology 40:663-671, 1996), Cassarino et al.
(Biochim.
Biophys. Acta 1362:77-86, 1997), Swerdlow et al. (Neurology 49:918-925, 1997),
Sheehan et al. (J. Neurochem. 68:1221-1233, 1997), and Sheehan et al. (J.
Neurosci.
17:4612-4622, 1997), all of these being hereby incorporated by reference.
The term "screening" refers to the use of the invention to identify agents
that impact the monitored intracellular process or condition in a negative or
positive
fashion. Cells or organelles are treated with an agent thought to impact the
monitored
intracellular process or condition, and the response of a subcellular
compartment of
interest to the agent is monitored and compared to a control sample that has
been treated
with only the vehicle used to deliver the agent. Agents that impact the
monitored
intracellular process or condition result in an altered response of the
subcellular
compartment of interest relative to the response in the control sample. In
certain
aspects of the invention, agents that act in a species-specific manner are
identified by
the screening methods of the invention.
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
38
subcellular compartment; or (iii) when one ET molecule (i.e., the ET donor or
the ET
acceptor) is localized to a subcellular compartment and the other ET molecule
(i.e., the
ET acceptor or the ET donor) transiently or otherwise associates with that
subcellular
compartment.
In situation (i), a change in the efficiency and/or rate of energy transfer
between the ET donor and acceptor molecules correlates with a change in a
condition or
the occurrence of a given process within the subcellular compartment of
interest. Non-
limiting examples of this aspect of the invention, described in greater detail
below,
include methods for assaying mitochondria) membrane potential (0'I') or pH
potential
(OpH), 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.
In situation (ii), a change in the rate of energy transfer between the ET
donor and acceptor molecules correlates with a process that influences the
cellular
membrane (e.g., alters the membrane potential) containing either the ET donor
or ET
acceptor molecule, and/or influences the subcellular compartment bounded by
the
cellular membrane, which compartment contains the other member (e.g., ET
acceptor or
donor molecule) of the ET molecule pair. Non-limiting examples of this aspect
of the
invention, described in more detail herein, include methods for monitoring the
mitochondria) pore transition (MPT) and viral uncoating processes.
In situation (iii), a change in the rate of energy transfer between the ET
donor and acceptor molecules correlates with the association of a detectably
labeled
molecule (e.g., labeled with either an ET donor or ET acceptor) with, or its
dissociation
from, a labeled subcellular compartment (e.g., labeled with either an ET
acceptor or ET
donor). Non-limiting examples of such embodiments of the invention, described
in
greater detail below, include methods for monitoring the association of Bcl-2
protein
with, or the dissociation of cytochrome c from, the outer mitochondria)
membrane.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
39
Donor-Acceptor Pairs
There are provided, according to the present invention, paired ET
molecules wherein each pair comprises an ET donor molecule and an ET acceptor
molecule. As described herein there are several criteria for determining
combinations
of energy-donating compounds (ET donor molecules) and energy-accepting
compounds
(ET acceptor molecules) that are acceptable for ET-based assays of the
invention.
Additional criteria may specifically apply when the assay is designed to
monitor a
particular intracellular state or activity such as, for example, mitochondrial
inner
membrane potential (Dyr or Dyrm), 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 absorption spectrum of
the ET
acceptor molecule, so that energy transfer from the donor to the acceptor can
occur.
Typically, an ET donor compound has an emission peak wavelength (herein,
"~,D(em)")
that is within several nm of the excitation peak wavelength of the acceptor
compound
(herein, "~,A(ex)"). That is, the difference between D(em) and Alex) is
typically from
about 70 nm to about 20 nm or less, with typical values for the difference
0 = 7~D(em) - ~.A(ex)
being <60 nm, <50 nm, <40 nm, <30 nm, <25 nm, <20 nm, <15 nm, <10 nm, <5 nm or
<1 nm. When excitation or emission is plotted as a function of wavelength,
however,
certain compounds that are suitable for use as ET donor molecules or ET
acceptor
molecules may have broad peaks, such that energy may be detectably transferred
between certain paired ET donor and ET acceptor molecules having a larger
difference
between D(em) and Alex) than that just described. For example, 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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
wavelength and/or the emission peak wavelength for the ET molecule), so long
as the
ET donor and the ET acceptor are within sufficient proximity of one another
for
detectable energy transfer to occur. Those having ordinary skill in the art
can readily
determine without undue experimentation when fluorescence resonance energy
transfer
5 is present, such that selection of appropriate ET donor-acceptor pairs may
be
accomplished according to established criteria and the teachings provided
herein.
For example, routine screening may be employed by combining in
solution (e.g., in the absence of a biological sample) at least a candidate ET
donor
molecule and a candidate ET acceptor molecule as disclosed herein, for
purposes of
10 determining whether a detectable FRET signal can be generated. For certain
donor-
acceptor combinations, selective accumulation of one or both of the donor and
acceptor
in a subcellular compartment may depend on binding of the donor and/or the
acceptor to
a molecule present in the subcellular compartment, and for other donor-
acceptor pairs
accumulation in such compartments may not involve such binding. Thus,
screening of
15 certain donor-acceptor pairs for their facilitation of a detectable FRET
signal in solution
may include adding to the solution at least one suitable biomolecule such as a
protein-
or peptide-, a lipid-, a nucleic acid- or a carbohydrate-containing species
that will be
selected by the person having ordinary skill in the art based upon familiarity
with the
nature of the donor and/or the acceptor and/or the properties of a subcellular
20 compartment in a contemplated biological sample to be used in the subject
invention
method. Without wishing to be bound by theory, in order to detect a FRET
signal the
concentrations of the ET donor and acceptor molecules used in such a pilot
experiment
may in certain such instances exceed those to be used in the subject invention
methods
as provided herein. However, similarly detectable concentrations of such ET
molecules
25 may accumulate in a sample subcellular compartment as described herein,
even where
substantially lower concentrations of ET molecules are initially contacted
with the
sample. Those familiar with the art will also readily appreciate that the
fluorescence
spectral properties of ET donor and ET acceptor molecules may vary as a
function of
solution and sample conditions employed (e.g., solvent selected, solvent and
ionic
30 strength, pH, nature of the sample, etc.).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
41
Another criterion useful in selecting a suitable ET donor-acceptor pair
for use according to the present invention is that the emission signal from
the excited
ET acceptor compound must be capable of being distinguished from the emission
signal
from the excited ET donor compound. An emission signal from an excited donor
can
be so distinguished if, for example, (1) the wavelength of the emission signal
from the
excited acceptor is sufficiently distinct from the wavelength of the emission
signal from
the excited donor or (2) the acceptor quenches the emission signal from the
excited
donor.
A variety of classes of compounds can serve as ET acceptor molecules
and ET donor molecules according to the present invention, and the acceptor
and donor
can, but need not, belong to the same class of compound. For instance, a
fluorescent
protein might serve as an ET donor molecule for an ET acceptor that is a small
organic
compound, or to an acceptor that is a different fluorescent protein, so long
as other
criteria necessary for the assay are satisfied. Table 1 lists, among other
things,
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).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
42
Table 2: Donor-Acceptor Pairs for ET-Based Assays
DONORS ACCEPTORS
Peak Peak Peak Peak
ExcitationEmission ExcitationEmission
Wave- Wave- Wave- Wave-
Compound length length length length Compound
Group I 373 - 400-500 Suitable
388 for Use
with Any
Group
I
nm nm Donor:
BFP- 380 nm 440 nm 433 nm 475 nm CFP-
F64L/S65T/ (501 F64L/S65T/
nm)*
Y66H/Y 145F Y66W/N I 46I/
M153T/
BFP-Y66H/ 381 nm 445 nm V 163A/N212L
Y145F
BFP-Y66H 382 nm 448 nm 461 nm 585 nm 2-Di-1-ASP
BFP-F64M/ 385 nm 450 nm 461 nm 589 nm DASPEI
Y66H/V68I
LysoTrackerTM373 nm 422 nm 470 nm* 510 nm wildtype
Yellow DND- GFP
22
LysoSensorTM 374 nm 424 nm 466 nm 536 nm NBD C6-
Yellow DND- ceramide
192
LysoSensorTM 373 nm 425 nm 466 nm 536 nm NBD C6-
Yellow DND- sphingomyelin
167
475 nm 605 nm 4-Di-1-ASP
442 nm 505 nm LysoSensorTM
Green DND-
153
443 nm 505 nm LysoSensorTM
Green DND-
189
479 nm 507 nm RFP-S65C
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
43
482 nm 504 nm DiOC7(3)
483 nm (none) SYTO~ 18
484 nm 501 nm DiOC6(3)
484 nm 500 nm DiOCS(3)
488 nm 507 nm RFP-
F64L/S65T
489 nm 511 nm RFP-S65T
490 nm 509 nm RFP-F64M/
S65G/Q69L
485 - 585 590 nm JC-1
nm aggregates*
Group IIA 360 - 465 -560Suitable Use with Any Group
375 for
nm nm or IIC Donor: IIA, IIB
DAPI 365 nm 520 nm 466 nm 536 nm rIBD C6-
ceramide
hydroxystilba-361 nm 536 nm 466 nm 536 nm NBD C6-
midine, sphingomyelin
methane-
sulfonate
Group IIB 390 - 465 -560475 nm 605 nm 4-Di-1-ASP
405
nm nm
wildtype 395 nm 510 nm 483 nm (none) SYTO~ 18
GFP (470 nm)*
484 nm 500 run DiOCS(3)
502 nm 512 nm YFP-
S65G/Y66W/
S72A/T203Y
503 nm 510 nm Brefeldin
A,
BODIPY~ FL
conj ugate
isomer 1
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
44
Group IIC 445 - 465 - 507 nm 529 nm rhodamine
460 560 123
nm nm
Lucigenin 455 nm 505 nm 510 nm 527 nm JC-1
monomers*
505 nm 511 nm BODIPY~ FL
CS-ceramide
505 nm 512 nm BODIPY~ FL
C5-
sphingomyelin
489 nm 520 nm acridine orange
504 nm 511 nm LysoTrackerTM
Green DND-26
508 nm (none) FL1N-1TM
532 nm 545 nm LysoTrackerTM
Green Br2
534 nm 551 nm LysoTrackerTM
Yellow DND-
68
541 nm 640 nm Neutral Red
528 nm 551 nm rhodamine
6G
524 nm 550 nm Tetrabromor-
hodamine 123
528 nm 551 nm rhodamine
6G
533 nm 545 nm BODIPY~ FL
Br2 CS_
ceramide
546 nm 590 nm ethidium
bromide
549 nm 565 nm DilC~g(3)
549 nm 565 nm Di1C16(3)
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
485 - 585 590 nm JC-1
nm aggregates*
*
559 nm 568 nm Brefeldin
A,
BODIPY FL
558/568
conjugate
isomer 1
Group III Suitable
425-440 450-535 for Use
with Any
Group
III
nm nm Donor:
CFP-F64L/ 433 nm 475 nm 461 nm 585 nm 2-Di-1-ASP
S65T/Y66W/ 501 nm*
N 146I/M153T/
V 163A/N212L 461 nm 589 nm DASPEI
466 nm 536 nm NBD C6-
ceramide
466 nm 536 nm NBD C6-
sphingomyelin
483 nm (none) SYTO~ 18
484 nm 500 nm DiOCs(3)
484 nm 501 nm DiOC6(3)
485 - 585 590 nm JC-1
nm aggregates
* *
489 nm 520 nm acridine orange
502 nm 512 nm YFP-
S65G/Y66W/
S72A/T203Y
503 nm 510 nm Brefeldin
A,
BODIPY~ FL
conjugate
isomer 1
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
46
504 nm 511 nm LysoSensorTM
Green DND-26
505 nm 511 nm BODIPY~ FL
CS-ceramide
505 nm 512 nm BODIPY~ FL
sphingomyelin
508 nm (none) FLJN-1TM
528 nm 551 nm rhodamine
6G
532 nm 545 nm LysoSensorTM
Green Br,
534 nm 551 nm LysoTrackerT"''
Yellow DND-
68
541 nm 640 nm Neutral red
Group IV 470 - 500 505 - Suitable Use with Any Group
565 for
nm nm Donor: IV
RFP-S65C 479 nm 507 nm 507 nm 529 nm rhodamine
123
RFP- 488 nm 507 nm 510 nm 527 nm JC-1
F64L/S65T monomers*
RFP-S65T 489 nm 511 nm 524 nm 550 nm tetrabromorhod
amine 123
MitoFluorTM 489 nm 517 nm 528 nm 551 nm rhodamine
6G
Green
RFP-F64M/ 490 nm 509 nm 548 nm 573 nm TMRM
S65G/Q69L
MitoTracker~490 nm 516 nm 549 nm 574 nm TMRE
Green FM
NAO 495 nm 519 nm 550 nm 574 nm tetramethylrosa
mine
wildtype 470 nm* 510 nm 556 nm 578 nm rhodamine
B
GFP ~
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
47
acridine 489 nm 520 nm 505 nm 511 nm BODIPY~ FL
orange
Cs-ceramide
505 nm 512 run BODIPY~ FL
Cs-
sphingomyelin
508 nm (none) FUN-1TM
533 nm 545 nm BODIPY~ FL
Br2 Cs_
ceramide
534 nm 551 nm LysoTrackerTM
Yellow DND-
68
541 nm 640 nm Neutral red
549 nm 565 nm DilC~g(3)
549 nm 565 nm DilC~6(3)
559 nm 568 nm Brefeldin
A,
BODIPY FL
558/568
conjugate
isomer 1
Group V 495 - 509 511 - Suitable
570 for Use
with Any
Group
V
nm nm Donor:
YFP-S65G/ 502 nm 512 nm 510 nm 527 nm JC-1
Y66W/S72A/ monomers*
T203Y 524 nm 550 run tetrabromorhod
amine 123
"FLASH" 508 nm 528 nm 528 nm 551 nm rhodamine
6G
proteins
533 nm 545 nm BODIPY~ FL
Br2 Cs_
ceramide
534 nm 551 nm LysoTrackerTM
Yellow DND-
68
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
48
541 nm 640 nm Neutral red
548 nm 573 nm TMRM
549 nm 574 nm TMRE
549 nm 565 nm DilC~g(3)
549 nm 565 nm DilC~6(3)
550 nm 574 nm tetramethylrosa
mine
556 nm 578 nm rhodamine
B
556 nm 585 nm 4-
dimethylamino-
tetramethyl-
rosamine
559 nm 568 nm Brefeldin
A,
BODIPY FL
558/568
conjugate
isomer 1
Group VI Suitable
545 - 560 for Use
565 - 625 with Any
Group
nm nm Donor:
VI
MitoTracker~551 576 579 nm 601 nm DiOC2(5)
Orange
CMTMRos 589 nm 617 nm BODIPY~ TR
ceramide
* Minor excitation or emission peak.
** JC-1 monomers vs. JC-1 aggregates: at higher concentrations (aqueous
solutions >
0.1 uM) or in mitochondria with higher potentials, and the "J-aggregates: have
different
spectral properties than the parent compound.
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
49
compounds preferentially accumulate in a water-based subcellular site or
compartment.
Some such compounds are known to preferentially accumulate at particular
subcellular
locations. Additionally or alternatively, a moiety that directs a compound to
a
subcellular location can be conjugated to a donor or acceptor moiety in order
to
generate a donor or acceptor compound capable of preferentially accumulating
at the
subcellular location of choice. For example, published PCT application WO
98/17826,
herein incorporated by reference, describes methods for conjugating
mitochondria-
directing moieties to various compounds.
Small lipophilic molecules, can be used when it is desired to have a
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.
Examples of proteins that can serve as donor and acceptor compounds
include fusion proteins comprising a "FLASH" (fluorescein arsenical helix
binder)
sequence (Griffin et al., Science 281:269-272, 1998), or an aequorin protein
or a green
fluorescent protein (Kendall et al., Trends in Biotechnology 16:216-224, 1998,
and
references cited therein). As used herein, the term "green fluorescent
protein"
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.
In order to generate an expression construct that produces an aequorin,
GFP or FLASH fusion protein that accumulates in the organelle or other
subcellular site
of interest, an expression vector comprising nucleotide sequences appropriate
for gene
expression can be manipulated to comprise (1) a first nucleic acid encoding a
GFP
derivative or FLASH polypeptide and (2) a second nucleic acid encoding a
peptide
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
sequence that directs a protein to an organelle or other subcellular site of
interest (i. e.,
the "targeting sequence"), wherein the first and second nucleic acids are
linked so as to
have a common reading frame that comprises both nucleic acids. Such fusion
proteins
can be directed to a particular membrane within a cell (such as, for example,
the nuclear
5 membrane or the inner or outer membrane of organelles such as mitochondria
and
chloroplasts), or to other specific subcellular locations, depending on the
nature of the
particular targeting sequence that is used in a given instance. Table 3 lists
some non-
limiting examples of intracellular sites wherein the donor and acceptor
compounds
listed in Table 2 accumulate.
10 Table 3: Sites of Localization of Non-Protein Donor and Acceptor
Compounds to Subcellular Compartments
Subcellular CompartmentCompounds
Endoplasmic reticulumBODIPY~ TR ceramide; DiOCS(3); NBD C6-ceramide;
&
Golgi apparatus
NBD C6-sphingomyelin; Brefeldin A; BODIPY~
FL
conjugate isomer 1; BODIPY~ FL C5-ceramide;
BODIPY~ FL CS- sphingomyelin; BODIPY~ FL
Br2 CS-
ceramide; DiIC ~ 8(3); and DiIC ~ 6(3)
Lysosomes & other acridine orange; FUN-1TM; hydroxystilbamidine,
acidic methane-
organelles sulfonate; LysoTrackerTMs Blue DND-22,
Green Br2,
Green DND-26, and Yellow DND-68; neutral
red;
LysoSensorTMS Blue DND-167, Blue DND-192,
and Green
DND-25 3
Mitochondria 2-Di-1-ASP; 4-Di-1-ASP; DASPEI; SYTO~ 18;
DiOC6(3); rhodamine 123; tetrabromorhodamine
123; JC-
l; ethidium bromide; rhodamine 6G; TMRM;
TMRE;
tetramethylrosamine; rhodamine B; 4-dimethylamino-
tetramethylrosamine; rhodamine 6G; DiOC2(5);
also
DiOC7(3) (plant mitochondria).
A further criterion is that the donor and acceptor compounds should
15 accumulate in the subcellular compartment at the same site, which will
permit ET to
take place, or at acceptably adjacent sites. By "acceptably adjacent" it is
meant that
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
51
such sites are within close enough proximity for ET to occur. Such sites are
from about
100 Angstroms (~) to about 10 ~ or less from each other, typically about 80 A,
60 ~,
50 A, 40 ~, 30 ~, 25 ~, 20 A, 15 ~, 10 ~, 5 ~ or less from each other,
preferably 70 ~
or less from each other, more preferably 50 ~ or less from each other, and
most
preferably 40 ~ or less from each other, depending on the donor-acceptor pair
of
compounds. In any event, because the relationship of (i) the distance between
an ET
donor molecule and an ET donor molecule to (ii) the ability for ET to
transpire is well
established (see, e.g., Haugland, 1996 Handbook of Fluorescent Probes and
Research
Chemicals- Sixth Ed., Molecular Probes, Eugene, OR). those familiar with the
art will
readily appreciate that donor-acceptor intermolecular distance is a cardinal
determinative factor for the efficiency of ET.
As a non-limiting example, one subcellular site of interest is the
organelle known as the mitochondrion. The mitochondrion comprises an outer
membrane that is exposed to the cytoplasm and with which various cytoplasmic
factors
may transiently or stably associate, an inner membrane, an intermembrane space
between the inner and outer membranes, and a matrix (the compartment within
the inner
membrane), arranged as is shown in Figure 1. For mitochondria, acceptably
adjacent
sites include (i) the outer membrane and the cytoplasm, including cytoplasmic
factors
associated with the outer membrane; (ii) the outer membrane and the
intermembrane
space; (iii) the intermembrane space and the inner membrane; and (iv) the
inner
membrane and the matrix, including factors within the matrix.
In the case of mitochondria, by way of example and not limitation, GFP
fusion protein derivatives have been targeted to the mitochondria) matrix
using
cytochrome c oxidase subunit IV protein sequences (Llopis et al., Proc. Nat).
Acad. Sci.
U.S.A. 95:6803-6808, 1993), to the intermembrane space using cytochrome c
protein
sequences (Mahajan et al., Nature Biotech. 16:547-552, 1998), and to the outer
membrane of mitochondria using hexokinase (Sui et al., Arch. Biochem. Biophys.
345:111-125, 1997), Bcl-2 or Bax (Mahajan et al., Nature Biotech. 16:547-552,
1998)
protein sequences. GFP fusion proteins have also been targeted to mitochondria
using
3-oxoacyl-CoA thiolase (Zhang et al., Biochem. Biophys. Res. Commun. 242:390-
395,
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
52
1998), OSCP (Prescott et al., FEBS Letts. 411:97-101, 1997) and BNIP3 (Yasuda
et al.,
J. Biol. Chem. 273:12415-12421, 1998) protein sequences. Aequorin fusion
protein
derivatives have been targeted to mitochondria using cytochrome c oxidase
protein
sequences (Proton et al., Biofactors 8:243-253, 1998; Rizzuto et al., Nature
358:325-
327, 1992). Other fusion proteins have been described that target
mitochondria) sites
using protein sequences from mitochondria) (or bacterial) thiolases (Arakawa
et al., J.
Biochem., Tokyo, 107:160-164, 1990), FO-ATPase subunit 9 (J. Biol. Chem.
271:25208-
25212, 1996), manganese superoxide dismutase (Balzan et al., Proc. Nat). Acad.
Sci.
U.S.A. 92:4219-4223, 1995), and P-450(SCC) (Kumamoto et al., J. Biochem.,
Tokyo,
10:72-78, 1989).
In the case of chloroplasts, by way of example and not limitation, fusion
proteins have been targeted to the outer membrane by use of the SCE70 heat
shock
protein targeting sequence (Wu et al., J. Biol. Chem. 268:19384-19391, 1993).
Other
targeting sequences, such as those from the Rieske iron-sulfiur protein
(Madueno et al.,
J. Biol. Chem. 269:17458-17463, 1994), direct fusion proteins across the
thylakoid
membrane.
If dual targeting to mitochondria and chloroplasts is desired, some fusion
proteins comprising dual targeting sequences have been described (Creissen et
al., Plant
J. 8:167-175, 1995; Huang et al., Plant Cell 2:1249-1260, 1990). Conversely,
when
plant cells are being used and targeting to only mitochondria or chloroplasts
is desired,
care must be taken to ensure that a dual targeting sequence is not employed.
In the case of the nucleus, by way of example and not limitation,
aequorin fusion protein derivatives have been targeted to the nucleus using
nucleoplasmin protein sequences (Badminton et al., J. Biol. Chem. 271:31210-
31214,
1997).
In the case of the endoplasmic reticulum (ER), by way of example and
not limitation, aequorin fusion protein derivatives have been targeted to the
endoplasmic reticulum using calreticulin protein sequences (Kendal) et al.,
Biochem.
Biophys. Res. Commun. 189:1008-1016, 1992).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
53
In the case of the Golgi apparatus, by way of example and not limitation,
aequorin fusion protein derivatives have been targeted to the Golgi plasma
membrane
using galactosyltransferase, SNAP-25, connexin and 5-HT1A-receptor protein
sequences
(Burton et al., Mol. Cell. Biol. 7:419-434, 1996; Marsault et al., EMBO J.
16:1575-
1581, 1997; Daguzan et al., Int. J. Dev. Biol. 39:653-657, 1995). GFP fusion
proteins
have been targeted to the Golgi apparatus using galactosyltransferase protein
sequences
(Llopis et al., Proc. Natl. Acad. Sci. U.S.A. 95:6803-6808, 1993)
In the case of whole cell assays, another criterion is that the
accumulation of ET donor and acceptor molecules should occur preferentially at
sites
within the mitochondrion or whichever organelle or subcellular compartment is
of
interest. However, some accumulation of the compounds in other, secondary
intracellular sites in acceptable, particularly if the donor and acceptor do
not accumulate
at the same secondary intracellular site (i.e., so that ET cannot occur in the
secondary
sites), or if the amount of background ET-derived signal is low enough that
events
specific to the organelle of interest can be followed despite accumulations)
of
compounds) at secondary sites. Moreover, most if not all of the assays
described
herein can be adapted for use with isolated organelles, in which instance
preferential
accumulation is not a criterion.
Instrumentation for Detecting Ener~yy 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
instruments) 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.
With regard to factors ( 1 ) and (2), the spectra of energy being applied to
samples to excite a donor compound, and the spectra of energy being emitted by
an
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
54
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 7~A(em), the minimal acceptable amount of difference between these two
values will
be influenced by, among other factors, the instrumentation being used. That
is, as
~,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. Conversely, an assay using a donor-acceptor pair
wherein
the difference between ~,D(em) and 7~A(em) is greater than about 50 to about
75 nm
requires an instrument of medium to low resolution.
With specific regard to factor (2), the type of energy being emitted by an
excited acceptor compound and measured in samples will determine, in general,
what
type of instrument will be used. By definition, a fluorometer is a device that
measures
fluorescent energy and should therefor be part of the instrumentation. A
fluorometer
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 fmaxT""
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.
With regard to factor (3), the type of samples to be assayed in a given
program, different formats will be appropriate for different types of samples.
For
example, 96-well microplates are suitable in instances where the cells or
isolated
organelles of interest adhere to the material of the microplate or to some
material
applied to the wells of the microplate; however, plastic fluorescence results
in a larger
background component at excitation wavelengths below about 400 nm. For
measurements involving nonadherent cells or organelles, or soluble extracts
prepared
therefrom, an instrument capable of reading fluorescent signals in glass or
polymeric
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
tubes or tubing is preferred. Regardless of what type of format is used, it
should allow
for the introduction of donor and acceptor compounds, as well as control
reagents and
compounds being evaluated, into the samples at appropriate points in time.
Factor (4), the number of samples to be assayed in a given program, will
5 influence how automated the instrument will be. For example, when high
throughput
(HTS) assaying of a large number of samples is desired, robotic or semi-
robotic
instruments are preferred. However, a fair number of samples can be processed
manually, particularly when formats that accommodate large sample numbers
(such as,
e.g., 96-well microplates) are used.
10 Depending on the assay, a Fluorometric Imaging Plate Reader (FLIPRT"')
instrument (Molecular Devices, Sunnyvale, CA) is often the instrument of
choice for
ET-based assays of the invention. The FLIPRT"' system (see
http://www.moleculardevices.com/pages/flipr.html) has the following desirable
features: it uses a combination of a water-cooled, argon-ion laser
illumination and
15 cooled CCD camera as an integrating detector that accumulates signal over
the period of
time in which it is exposed to the image and, as a result, its signal-to-noise
characteristics are generally superior to those of conventional imaging
optics; it also
makes use of a proprietary cell-layer isolation optics that allow signal
discrimination on
a cell monolayer, thus reducing undesirable extracellular background
fluorescence; it
20 provides data in real-time, and can also provide kinetic data (i.e.,
readings at a multitude
of timepoints); it has the ability to simultaneously stimulate and read all 96
wells of a
96-well microplate; it provides for precise control of temperature and
humidity of
samples during analysis; it includes an integrated state-of the-art 96-well
pipettor,
which uses dispensable tips to eliminate carryover between experiments, that
can be
25 used to aspirate, dispense and mix precise volumes of fluids from
microplates; and, in
the case of the FLIPR3g4 instrument, it can be adapted to run sample assays in
a robotic
or semi-robotic fashion, thus providing for analysis of large numbers of
samples in
shortest amount of time (e.g., up to about a hundred 96-well microplates per
day).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
56
Monitoring Conditions or Processes within Subcellular Compartments
The term "subcellular compartment" refers to any intracellular space that
is, for at least some of the time, maintained in an at least partially
isolated condition.
Some type of physical barrier, typically a bilipid membrane, forms the border
between a
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. Some of the subcellular compartments that can be monitored or
assayed
using the present invention, and applications particular for each such
subcellular
compartment, are described in more detail in the following subsections.
T ~f:f,~..l......a..; .,
One subcellular compartment of particular interest is the organelle
known as the mitochondrion (plural, mitochondria). Mitochondria are the main
energy
source in cells of higher organisms, and provide direct and indirect
biochemical
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 mitochondria) role in intracellular
calcium
homeostasis.
In addition to their role in energy production in growing cells,
mitochondria (or, at least, mitochondria) components) participate in
programmed cell
death (PCD), also known as apoptosis (Newmeyer et al., 1994, Cell 79:353-364;
Liu et
al., 1996, Cell 86:147-157). Apoptosis is apparently required for normal
development
of the nervous system and functioning of the immune system. Moreover, some
disease
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
57
states are thought to be associated with either insufficient or excessive
levels of
apoptosis (e.g., cancer and autoimmune diseases in the first instance, and
stroke damage
and neurodegeneration in Alzheimer's disease in the latter case). For general
reviews of
apoptosis, and the role of mitochondria therein, see Green and Reed (1998,
Science
281:1309-1312), Green (1998, Cell 94:695-698) and Kromer (1997, Nature
Medicine
3:614-620). Thus, agents that affect apoptotic events, including those
associated with
mitochondrial components, might have a variety of remedial, therapeutic,
palliative,
rehabilitative, preventative, prophylactic or disease-impeditive uses.
A variety of apoptogens are known to those familiar with the art (see,
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-
a,); Fas
ligand; glutamate; N-methyl-D-aspartate (NMDA); interleukin-3 (IL-3);
herbimycin A
(Mancini et al., 1997 J. Cell. Biol. 138:449-469); paraquat (Costantini et
al., 1995
Toxicology 99:1-2); ethylene glycols; protein kinase inhibitors, such as,
e.g.,
staurosporine, calphostin C, caffeic acid phenethyl ester, chelerythrine
chloride,
genistein; 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; N-[2-((p-
bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide; KN-93; quercitin; d-
erythro-sphingosine derivatives; UV irradiation; ionophores such as, e.g.:
ionomycin
and valinomycin; MAP kinase inducers such as, e.g.: anisomycin, anandamine;
cell
cycle Mockers 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.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
58
Sci. USA 95:4997-5002 and references cited therein); calcium and inorganic
phosphate
(Kroemer et al., 1998 Ann. Rev. Physiol. 60:619).
Mitochondria) ultrastructural characterization reveals the presence of an
outer mitochondria) membrane that serves as an interface between the organelle
and the
cytosol, a highly folded inner mitochondria) membrane that appears to form
attachments
to the outer membrane at multiple sites, and an intermembrane space between
the two
mitochondria) membranes (see Figure 2). The subcompartment within the inner
mitochondria) membrane is commonly referred to as the mitochondria) matrix.
(For a
review, see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s.) The cristae,
originally
postulated to occur as infoldings of the inner mitochondria) membrane, have
recently
been characterized using three-dimensional electron tomography as also
including tube-
like conduits that may form networks, and that can be connected to the inner
membrane
by open, circular (30 nm diameter) junctions (Perkins et al., 1997, Journal of
Structural
Biology 119:260). While the outer membrane is freely permeable to ionic and
non-ionic
solutes having molecular weights less than about ten kilodaltons, the inner
mitochondria) membrane exhibits selective and regulated permeability for many
small
molecules. including certain cations, and is impermeable to large (> -~-10
kDa)
molecules.
Four of the five multisubunit protein complexes (Complexes I, III, IV
and V) that mediate ETC activity are localized to the inner mitochondria)
membrane.
The remaining ETC complex (Complex II) is situated in the matrix. In at least
three
distinct chemical reactions known to take place within the ETC, protons are
moved
from the mitochondria) matrix, across the inner membrane, to the intermembrane
space.
This disequilibrium of charged species creates an electrochemical potential of
approximately 220 mV referred to as the "protonmotive force" (PMF), which is
often
represented by the notation O~I' or 0'1'm. O~Ym represents the sum of the
electric
potential and the pH potential (i.e., the pH differential) across the inner
mitochondria)
membrane (see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s and references
cited
therein).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
59
D~fm 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. ~~fm is also the driving force for
the influx
of cytosolic Ca2+ into the mitochondrion. Under normal metabolic conditions,
the inner
membrane is impermeable to proton movement from the intermembrane space into
the
matrix, leaving ETC Complex V as the sole means whereby protons can return to
the
matrix. When, however, the integrity of the inner mitochondria) membrane is
compromised, as occurs during mitochondria) permeability transition (MPT) that
accompanies certain diseases associated with altered mitochondria) function,
protons
are able to bypass the conduit of Complex V without generating ATP, thereby
uncoupling respiration from energy production. During MPT, 0'fm collapses and
mitochondria) membranes lose the ability to maintain an equilibrium
distribution of one
or more ionic species or other solutes, i.e., to selectively regulate
permeability to solutes
small (e.g., ionic Ca2+, Na;, K+, H+) and/or large (e.g., proteins).
Loss of mitochondria) membrane electrochemical potential may be the
result of mechanisms such as free radical oxidation, or may be due to direct
or indirect
effects of mitochondria) and/or extramitochondrial gene products. Loss of
mitochondria) potential appears to be a critical event in the progression of
diseases
associated with altered mitochondria) function, including degenerative
diseases such as
Alzheimer's Disease; diabetes mellitus; Parkinson's Disease; Huntington's
disease;
dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondria)
encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis;
hyperproliferative disorders; mitochondria) diabetes and deafness (MIDD) and
myoclonic epilepsy ragged red fiber syndrome. To provide improved therapies
for such
diseases, agents that limit or prevent loss of mitochondria) membrane
potential (Dym)
may be beneficial. The present invention provides a novel approach to the
identification of agents useful for such diseases. The invention fulfills the
need for an
assay that permits rapid screening for agents capable of altering
mitochondria)
membrane potential and provides other related advantages.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
Assays for Measuring Changes in Parameters in Subcellular Compartments
When the ET-based assay is designed to measure a change in state of, or
decrease or increase in some activity at, a subcellular compartment or site,
such as 0~
of mitochondria or the presence or absence of factors that are transiently
associated with
5 or released from an intracellular site, an additional criterion for donor-
acceptor
compounds is that one of the compounds (either the donor or the acceptor
compound)
must accumulate in and/or be released from the subcellular compartment or site
in a
manner that is dependent on the chosen parameter or activity, whereas the
presence of
the other compound (the acceptor or donor, respectively) in the subcellular
10 compartment must be independent of the chosen parameter or activity.
Compounds whose mitochondria) concentration is dependent on Ayr
include, by way of example and not limitation, TMRM (Farkas et al., Biophys.
J.
56:1053-1069, 1989), TMRE (Ehrenberg et al., Biophys. J. X3:785-794, 1988),
rhodamine 123 (Scaduto et al., Biophys. J. 76:469-477, 1999), ethidium bromide
15 (Coppey-Moisan et al., Biophys. J. 71:2319-2328, 1996), DASPMI (4-Di-1-ASP
and 2-
Di-1-ASP) and DASPEI (Rafael et al., FEBS Lett. 170:181-185, 1984). Compounds
whose mitochondria) concentration is not dependent on Dyr include, by way of
example
and not limitation, NAO (Maftah et al., Biophys. Res. Commun. 164:185-190,
1989),
MitoTracker~ Green FM and MitoFluorTM Green (Haugland, Handbook of Fluorescent
20 Probes and Research Chemicals, 6th Ed., Molecular Probes, Inc., Eugene, OR,
1996, p.
269), and DAPI (Coppey-Moisan et al., Biophys. J. 71:2319-2328, 1996). Both
collapse and dissipation of Dyr can be monitored using such compounds. As used
herein, "0y collapse" refers to the rapid dissolution of Dyr, i. e., Dyr
reaches zero within
a few minutes after mitochondria are treated with an agent that induces
collapse of
25 mitochondria) membrane potential, such as, for instance CCCP or FCCP or any
other
agent capable of rapidly driving Dym to zero. The term "Dyr dissipation"
refers to a
slower decrease in Dyr that does not result in Dyr 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
mitochondria)
30 membrane potential, such as, for example, ionomycin, thapsigargin,
atractyloside,
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
61
A23187, 4-bromo-A23187, adenine nucleotide translocator inhibitors, inhibitors
of
mitochondria) 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. Those having
ordinary skill
in the art are familiar with any number of mitochondria) ETC inhibitors that
have been
characterized with regard to which ETC components may be impaired. For
additional
disclosure relating to measurement of mitochondria) membrane potential, agents
that
induce collapse of mitochondria) membrane potential and agents that induce
dissipation
of mitochondria) membrane potential, see U.S. Application Serial Nos.
09/161,172 and
09/185,904.
Using mitochondria as an example, a variety of factors are known to be
either ( 1 ) transiently associated with the outer membrane of the
mitochondrion or (2)
typically located at an intramitochondrial site but released from mitochondria
during
events such as, e.g.. mitochondria) pore transition (MPT) or apoptosis (a.k.a.
programmed cell death, PCD; for a review, see Green et al., Science 281:1309-
1312,
1998). Examples of proteins belonging to class ( 1 ) include hexokinase II,
and Bcl-2,
Bcl-XL, Bax and other members of the bcl-2 gene family (Kroemer, Nature Med.
3:614-
620, 1997; Nartita et al., Proc. Nat). Acad. Sci. U.S.A. 95:14681-14686,
1998).
Examples of class (2) factors that are released during MPT or apoptosis
include
cytochrome c (Yang et al., Science 275:1129-1132, 1997; Kluck et al., Science
275:1132-1136, 1997), procaspase-2 and -9 (Susin et al., J. Exp. Med. 189:381-
394,
1998) and apoptosis inducing factor (AIF; Susin et al., J. Exp. Med. 184:1331-
1341,
1996; Susin et al., J. Exp. Med. 186:25-37, 1997). 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. For example, hexokinase II fusion proteins that associate
with the outer
membrane of mitochondria (Sui et al., Arch. Biochem. Biophys. 345:111-125,
1997),
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
62
and cytochrome c fusion proteins that localize GFP (Mahajan et al., Nature
Biotech.
16:547-552, 1998) or other proteins (Nye et al., Mol. Cell. Biol. 10:5763-
5771, 1990) to
the intermembrane space of mitochondria, have been described. FLASH, aequorin
and
green fluorescent fusion proteins are used as donor or acceptor compounds in
FRET-
S based assays designed to monitor the degree and/or rate of mitochondria)
association or
release of factors having various biological functions.
The ET-based methods of the invention possess certain advantages over
other methods for assaying ~yrt". For example, methods that utilize a single
potentiometric fluorophore (i.e., a fluorophore that accumulates in
mitochondria in a
Dyrr"-dependent manner) may require that the fluorophores be present at
concentrations
that are toxic when agents that impact ~yfm are introduced (see, e.g., U.S.
Patent No.
5,169,788). In contrast, the ET-based assays of DyJn~ of the invention can be
carried out
using lower, non-toxic doses of fluorophores. Furthermore, plasma membrane
potential
contributes to the signal in assays where a single potentiometric flu~rophore
is used,
whereas the ET-based assays of the invention are specific for changes in
mitochondria)
membrane potential.
The detected fluorescence emission is typically compared to a reference
signal. For quantitative measurements of O~m, the reference signal may be the
signal
observed in mitochondria with a known 0'Pm, and one or more such references
signals
may be used. Alternatively, 0'Ym may be evaluated relative to a OIYm 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 O~fm, or
relative to a
O~fm 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.
Chloroplasts
The chloroplast is an organelle found in plant cells wherein
photosynthesis takes place. Photosynthesis, in addition to being an integral
part of a
plant cell's metabolism, is an important process that impacts many other
living
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
63
organisms as well. The reason for this is twofold: photosynthesis "fixes"
atmospheric
COZ into biologically usable carbohydrate (CHO)~ molecules and also produces
OZ
which is required by all aerobic organisms.
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
I S of nuclear DNA (Dope et al., J. Cell. Biol. 79:631-636, 1978).
The Nucleus and the Nucleolus
The nucleus is the organelle that comprises most (from the standpoint of
information, if not mass) of a cell's DNA in the form of several chromosomes
(Mitochondria and chloroplasts have their own DNA molecules that are typically
much
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
64
DNA, it appears that it is mainly ribosomal RNA (rRNA) molecules that are
produced
in the nucleolus.
Endosomes, Lysosomes and Peroxisomes
Cells assimilate extracellular fluid, and macromolecules dissolved
therein, by a process called endocytosis. Endocytotic vesicles are formed when
a
portion of the cell membrane evolves from a cup-shaped surface feature into an
inwardly-directed "bud" and, eventually, a small membrane-bound vesicle that
is taken
up into the cytosol. At least two mechanisms have been proposed for the
formation of
the cup-shaped surface features from which endosomes originate. First, local
changes
in the structure and/or composition of the lipid bilayer portion of the cell
membrane can
induce membrane curvature over a limited area thereof. Second, one or more
coat
proteins can act on a given location in the cell membrane to induce the
formation of a
cup-shaped surface feature. In the latter instance, the most well-
characterized example
are the "coated pits" that are formed, at least in part, by the protein
clathrin (for a
review, see Schekamn and Orci, Science 271:1526-1533, 1996).
Lysosomes contain various hydrolytic enzymes, each of which catalyzes
the breakdown of specific types of macromolecules. Primary lysosomes
containing
such enzymes are produced intracellularly and may fuse with endosomes to form
secondary lysosomes. In the latter type of vesicle, the enzymes from the
primary
lysosome are brought into contact with, and are thus free to act upon, the
contents of the
endosome. In general, after enzymatic digestion of the contents of the
secondary
lysosome, its membrane is dissolved in order to release its contents into the
cytosol.
The formation and fate of, e.g., secondary lysosomes can be followed
using the methods of the invention in the following manner. Cells are
engineered to
produce one or more lysosomal enzymes modified to contain a moiety capable of
serving as an acceptor or donor in energy transfer. Such cells are brought
into contact
with an agent that is taken up in endosomes, wherein the agent is or has been
modified
to be an ET acceptor or donor, respectively. When the resultant endosomes fuse
with a
primary lysosome, the acceptor and donor are present in the same subcellular
CA 02375542 2001-12-13
WO 00/79274 _ PCT/US00/17380
compartment (the secondary lysosome), and ET occurs and can be monitored as
described herein. The dissolution of the secondary lysosome liberates the ET
acceptor-
donor pair of molecules, which are then separated from each other as they are
diluted
into the cytosol, wherein the degree of ET decreases or ceases altogether.
5 Peroxisomes are another type of intracellular vesicles bounded by a
single membrane. Unlike lysosomes, which generally contain hydrolytic enzymes,
peroxisomes contain oxidative enzymes that generate and destroy hydrogen
peroxide.
Endoplasmic Reticulum
The endoplasmic reticulum (ER) is composed of a series of flattened
10 sheets, tubes and sacs that enclose a large intracellular space. The
membrane of the ER
is in structural continuity with the outer nuclear membrane and extends
throughout the
cytoplasm. Some functions of the ER include the synthesis and transport of
membrane
proteins and lipids. Generally speaking, two types of ERs may exist in a cell.
Smooth
ER is generally tubular in shape and is typically devoid of attached
ribosomes; one
15 major function of smooth ER is lipid metabolism. Rough ER typically occurs
as
flattened sheets, the cytosolic side of which is usually associated with many
active
(protein-synthesizing) ribosomes.
Gobi Apparatus
The Golgi apparatus is a system of stacked, flattened and membrane
20 enclosed sacs and is generally thought to be involved in the modification,
sorting and
packaging of macromolecules for secretion or for delivery to other subcellular
compartments. Numerous small (> ~50 nM) membrane-enclosed vesicles are thought
to comprise macromolecules in order to carry out the transport thereof between
the
Golgi apparatus and other subcellular compartments.
25 Subor~anellar Compartments
Certain components of organelles are also subcellular compartments
within the scope of the invention. For example, mitochondria, chloroplasts and
nuclei
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
66
are surrounded by two membranes. The space between a set of paired membranes
is not
itself an organelle, but is a subcellular compartment as defined herein. Such
spaces are
named, e.g., and respectively, the mitochondria) 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. In this
aspect of the invention, membrane-directed donors [such as, e.g., 9-
anthrylvinyl
(LAPC)] and acceptors such as 3-perylenoyl (LPPC) are incorporated into one or
more
membranes of choice. The partition coefficients between membrane and aqueous
phases are 8.3 x 10' and 10.5 x 10' for LAPC and LPPC, respectively (Razinkov
et al.,
Biochim. Biophys. Acta 1329:149-158, 1997).
I 5 Intracellular Parasites
Other subcellular compartments of interest include intracellular parasites
such as viruses and intracellular bacteria such as Rickettsiae and Chlamydia
spp.
Viruses consist of a genome, which may be composed of either DNA or RNA, that
is
surrounded by a protein shell. In the case of animal viruses, this protein
shell is often
itself enclosed within an envelope comprising both protein and lipid. Viruses
multiply
only within cells, as they are dependent on the host cells' macromolecular
synthetic
processes. They have thus been described as "genetic parasites."
One example of how the present invention may be applied to such
intracellular parasites, provided by way of illustration and not limitation,
is as follows.
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
67
"uncoating" and typically takes place in the cytoplasm (or a subcellular
compartment,
such as a vacuole, within the cytoplasm). Most animal viruses undergo
uncoating as a
result of the action of intracellular proteases on polypeptides that are a
part of the
capsid. Agents that inhibit or block viral uncoating, for example by
inhibiting the
action of intracellular proteases, are expected to be novel antiviral agents;
a method of
assaying viral uncoating would be useful for screening for such agents.
The present invention provides such a method for assaying viral
uncoating in, for example, the following manner. Viral particles are prepared
that
contain an acceptor-donor pair of molecules ("loaded viruses"); this can be
accomplished by, e.g., contacting viral particles or cells infected with
viruses with a
donor-acceptor pair of molecules that specifically localize to lipid
membranes. By way
of example and not limitation, the donor can be 9-anthrylvinyl (LAPC) and the
acceptor
can be 3-perylenoyl (LPPC) (Razinkov et al., Biochim. Biophys. Acta 1329:149-
158,
1997).
Viral adsorption typically occurs equally well at 4°C and
37°C, whereas
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
68
group grow both in the cytoplasm and the nucleus, and C. burnetii grows within
the
cytoplasm and phagolysosomes.
Chlamydiaceae is a family of obligate intracellular bacterial parasites
that infect a number of vertebrate hosts, typically birds or mammals
(including
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.
When elementary bodies are labeled with a donor or acceptor molecule, and
lysosomes
with an acceptor or donor molecule, respectively, energy transfer will occur
if
phagolysosomes are formed. Agents that inhibit the elementary body's ability
to
prevent fusion of phagosomes and lysosomes will result in energy transfer that
can be
monitored by the present invention; such agents are expected to be novel
antibiotics
useful for treating Chlamydia infections.
Assaying Interactions Between Macromolecules within or Associated with
Subcellular
Compartments
In another aspect of the invention, energy transfer is used to monitor
interactions between pairs of macromolecules found within or associated with
subcellular compartments. This embodiment, which is drawn to means for
monitoring
the association of a macromoleeular 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.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
69
As one example, some cancer cells are thought to result, at least in part
form overexpression of a protein that may preferentially associate with one or
more
subcellular compartments. The bcl-2 gene was initially identified as a causal
factor in
certain types of lymphatic cancers (B-cell lymphoma, hence the name) in which
bcl-2 is
overexpressed, resulting in an abnormally longer lifespan for B-cells, which
in turn is
thought to allow these cells to accumulate additional mutations resulting in
frank
malignancy and lymphatic tumor development (for reviews of the Bcl-2 family of
proteins, see Davies, Trends in Neuroscience 18:355-358, 1995; Kroemer, Nature
Med.
3:614-620, 1997; W095/13292; W095/00160; and U.S. Pat. No. 5,015,568).
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 mitochondria) membrane, the nuclear membrane and the
endoplasmic reticulum. Another member of the Bcl-2 family of proteins, Bax,
localizes
to the outer mitochondria) membrane. Although FRET has been used to
demonstrate
IS the interaction of Bcl-2 and Bax in individual mitochondria (Mahajan et
al., Nat.
Biotechnol. 16:547-552, 1998), energy transfer has not been used to monitor
the
association (or dissociation) of such proteins with (or from) subcellular
compartments.
The present invention provides methods for monitoring the interactions of
macromolecules with subcellular compartments.
One example of such a method is as follows. The width of the combined
inner and outer mitochondria membranes has been estimated to be 22 + 4 nm
(Perkins
et al., J. Structural Biol. 119:260-272, 1997). Accordingly, loading the
intermembrane
space with donor (or acceptor) molecules would be expected to bring them in
sufficiently close proximity with acceptor (or donor) molecules present within
or
associated with the outer mitochondria) membrane. Events such as the
localization of
Bcl-2 proteins to the outer mitochondria) membrane could thus be monitored by
tagging
Bcl-2 with an acceptor (or donor) that undergoes energy transfer with a donor
(or
acceptor) that has been loaded into the intermembrane space. In like fashion,
the
dissociation of proteins such as cytochrome c from mitochondria can be
followed using
donor- or acceptor-tagged cytochrome c proteins and acceptor- or donor-loaded
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
(respectively) intramembrane spaces. Such processes are thought to represent
significant events in apoptotic pathways (Green et al., Science 281:1309-1312,
1998;
Green, Cell 94:695-698, 1998).
Screening for Species-Specific Agents
5 In certain embodiments, the present invention provides screening assays
for identifying species-specific agents. A "species-specific agent" refers to
an agent
that affects a subcellular compartment of a first organism belonging to one
species but
that does not affect the homologous subcellular compartment of a second
organism
belonging to another species. Thus the invention provides a method for
identifying an
10 agent that preferentially alters a cellular membrane potential in a
subcellular
compartment of a first biological source without substantially altering a
corresponding
cellular membrane potential in a subcellular compartment of a second
biological source.
In preferred embodiments, the subcellular compartment is a mitochondrion and
the
cellular membrane potential is mitochondria) membrane potential. The screening
15 assays provided by the instant methods are thus directed in pertinent part
to assaying, in
the absence and presence of a candidate agent, a cellular membrane potential
by
contacting each of a first and second sample comprising one or more cellular
membranes from a first and a second distinct biological source, respectively,
with an ET
donor and an ET acceptor molecule, exciting the ET donor to produce an excited
ET
20 donor molecule, detecting a signal generated by energy transfer from the ET
donor to
the ET acceptor and comparing the signal generated in the absence of the
candidate
agent to the signal generated in the presence of the candidate agent.
In those certain preferred embodiments wherein the invention is directed
to a method for identifying an agent that preferentially alters mitochondria)
membrane
25 potential in mitochondria from a first biological source without
substantially altering
mitochondria) 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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
71
sites as provided herein. Typically, based upon the teachings provided herein,
a person
having ordinary skill in the art can readily determine when a candidate agent
alters a
cellular membrane potential such as mitochondria) 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 mitochondria) membrane potential are also provided in U.S.
application
number 09/ 161,172.
As used herein, an agent identified according to the instant method that
is a species-specific agent or an agent that "preferentially" alters
mitochondria)
membrane potential in the mitochondria from a first biological source (e.g., a
first
species) without substantially altering the mitochondria) 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
I S the first or the second species but not both) while effecting the death or
growth
impairment of the mitochondria or cells from the other species. Similarly,
where such
an agent does not "substantially" alter mitochondria) 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. Thus, preferential alteration of
mitochondria) membrane potential by such an agent may increase or may decrease
D~f""
as long as the effect is species-specific. Without wishing to be bound by
theory, 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.
In certain other embodiments an agent identified according to the instant
method that that "preferentially" alters mitochondria) membrane potential in
the
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
72
mitochondria from a first biological sample (e.g., a first tissue) without
substantially
altering the mitochondria) 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. Similarly, where such an
agent does
not "substantially" alter mitochondria) 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. Thus, preferential alteration of mitochondria) membrane potential by
such an
agent may increase or may decrease 0~f"" as long as the effect is sample-
specific.
According to these embodiments, an agent may be identified that acts
selectively in a
tissue-specific manner, such that the agent may be employed to manipulate
mitochondria) membrane potential in certain tissue types but not other, even
within the
same organism. Alternatively, the first and second tissues may be derived from
distinct
subjects of the same species, or from subjects of distinct species. For
example,
according to such a method of the instant invention, an agent may be
identified using
this approach that preferentially alters neuronal cell mitochondria) membrane
potential
without substantially altering liver cell mitochondria) membrane potential.
Using mitochondria as an example of a subcellular compartment, this
embodiment of the invention may be used, for example, to identify agents that
selectively induce collapse of Dy in mitochondria derived from different
species, e.g.,
in trypanasomes (Ashkenazi et al., Science 281:1305-1308, 1998), and other
eukaryotic
pathogens and parasites, including but not limited to insects, but which do
not induce
Dy collapse in the mitochondria found in the cells of their mammalian hosts.
Such
agents are expected to be useful for the prophylactic or therapeutic
management of such
pathogens and parasites.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
73
For example, members of the phylum Apicomplexa (formerly called
Sporozoa) comprise a large and diverse group of pathogenic protozoa that are
intracellular parasites. Some members, including species of Babesia, Theileria
and
Eimeria, cause economically important animal diseases, and other members, such
as
Toxoplasma gondii and Cryptosporidium spp. also cause human disease,
particularly in
immunocompromised individuals. The acomplexicans are unusual in terms of their
extrachromosomal DNA elements, as they comprise both a mitochondria) genome
and a
putative plastid genome (see Feagin, Annu. Rev. Microbiol. -18:81-104, 1994,
for a
review). Probably the most well-studied acomplexicans are species of
Plasmodium,
which cause malaria. Antimalarial agents include agents that specifically
impact the
function of Plasmodium mitochondria (Peters et al., <Ann. Trop. Med. Parsitol.
78:567-
579, 1984; Basco et al., J. Eukaryot. Microbiol. 41:179-183, 1994), and one
such agent,
atovaquone, collapses Dy in mitochondria from Plasmodium yoelii but has no
effect on
4~ of mammalian mitochondria (Srivastava et al., J. Biol. Chem. 2?2:3961-3966,
1997). Accordingly, the ET-based assay of Dyr of the present invention can be
used to
screen libraries of compounds for novel antimalarial agents, i.e., compounds
that cause
Dyr collapse in Plasmodium mitochondria but not in mammalian mitochondria.
As another example, this embodiment of the invention is used to create
and identify agents that selectively induce Dye 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. Such
agents are expected to be useful for the management and control of such
undesirable
plants and insects. Cultured insect cells, including for example, the Sft7 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.
In this embodiment of the invention, the subcellular compartment of
interest of a first species is loaded with a first donor-acceptor pair of
molecules which
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
74
fluoresce at a first wavelength, and the corresponding subcellular compartment
from a
second species is loaded with a second donor-acceptor pair of molecules which
fluoresce at a second wavelength. For example, mitochondria from two different
species may be loaded with such donor-acceptor pairs of molecules. The two
types of
loaded mitochondria are placed in a single chamber, and an agent to be tested
for its
ability to induce MPT in a species-specific manner is then also introduced
into the
chamber. The change in fluorescence at both the first and second wavelength is
measured over time in a concomitant fashion. For example, a Fluorometric
Imaging
Plate Reader (FLIPRT"') instrument (see infra) 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. 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.
Di~,nostics and Screening for Therapeutic A
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 mitochondria) membrane potential. In certain other preferred
embodiments the
invention provides a method for identifying a regulator of an agent that
alters
mitochondria) 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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
candidate agent or a candidate regulator, a cellular membrane potential by
contacting a
sample comprising one or more cellular membranes with an ET donor and an ET
acceptor molecule, exciting the ET donor to produce an excited ET donor
molecule,
detecting a signal generated by energy transfer from the ET donor to the ET
acceptor
5 and comparing the signal generated in the absence of the candidate agent (or
regulator)
to the signal generated in the presence of the candidate agent (or regulator).
Embodiments that are directed to a method for identifying a regulator of an
agent that
alters mitochondria) membrane potential further comprise contacting a sample,
prior to
the step of detecting, with an agent that is either a known agent that alters
mitochondria)
10 membrane potential or an agent that alters mitochondria) membrane potential
and that is
identified according to the methods provided herein.
In those certain preferred embodiments wherein the invention is directed
to a method for identifying an agent that alters mitochondria) membrane
potential, or to
a method for identifying a regulator of an agent that alters mitochondria)
membrane
15 potential, neither the ET donor molecule nor the ET acceptor molecule is
endogenous to
mitochondria, and the ET donor and the ET acceptor each localize independently
of one
another to the same submitochondrial site or to acceptably adjacent
submitochondrial
sites as provided herein. Typically, based upon the teachings provided herein,
a person
having ordinary skill in the art can readily determine when a candidate agent
alters a
20 cellular membrane potential such as mitochondria) 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 mitochondria) membrane potential are also provided in U.S.
application
number 09/ 161,172.
25 Similarly, for purposes of determining whether a compound that is a
candidate regulator of an agent that alters a cellular membrane potential such
as
mitochondria) membrane potential, methods for quantifying membrane potential
will be
useful. Agents that alter mitochondria) membrane potential include agents
known to
have such properties, including agents that dissipate mitochondria) membrane
potential
30 and agents that collapse mitochondria) membrane potential (e.g., those
described in
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
76
greater detail in the Examples below), as well as agents identified according
to methods
provided herein. A regulator of an agent that alters mitochondria) 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 mitochondria)
membrane
potential to alter mitochondria) membrane potential. Thus, for example, a
regulator of
an agent that alters mitochondria) membrane potential may be an agonist or may
be an
antagonist of the agent that alters mitochondria) membrane potential. For
example,
where an agent that alters mitochondria) membrane potential dissipates the
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 mitochondria)
membrane potential
may confer a protective effect on mitochondria) membrane potential when the
dissipating agent is present. Conversely, for an agent that alters
mitochondria)
membrane potential by preserving or enhancing 0~f"" regulators that are
agonists may
also protect or enhance potential while regulators that are antagonists may
lead to
dissipation or collapse of 0~'m. Without wishing to be bound by theory, a
regulator as
described herein may participate in intermolecular interaction events (e.g.,
recognition,
binding, complex formation, covalent modification, alteration of conformation)
with
one or more of an agent that alters mitochondria) membrane potential and the
subcellular target or targets of the agent that alters mitochondria) membrane
potential,
including mitochondria) molecular components. (Mitochondria) molecular
components
are described, for example, in U.S. application number 09/161,172.)
Thus, where a number of disorders and diseases result from processes
involving mitochondria, the main energy source in cells of higher organisms,
the
invention provides compositions and methods for monitoring mitochondria)
membrane
potential (Dyr) and changes therein via energy transfer, as noted above. As
described in
detail herein, Dyr is required for a variety of mitochondria) functions, and
defects in the
production or maintenance of Dyr are associated with many diseases and
disorders.
Furthermore, changes in Dy occur in a variety of subcellular processes that
can serve as
targets for the development of therapeutic agents. Thus, the ET-based assay of
Dy can
be used to help confirm the presence of a disease or disorder associated with
alterations
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
77
in Dyr 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)
Dye and can
thus be used to treat such diseases and disorders. Moreover, the ET-based
assay of Ayr
can be used to screen for agents that selectively perturb Dy 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. 91:227s-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
mitochondria) role in intracellular calcium homeostasis. In addition to their
role in
metabolic processes, mitochondria are also involved in the genetically
programmed cell
suicide sequence known as "apoptosis" (Green and Reed, Science 281:1309-1312,
1998; Susin et al., Biochim. et Biophys. Actu 1366:151-165, 1998).
Defective mitochondria) activity, including but not limited to failure at
any step of the elaborate mufti-complex mitochondria) 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, mitochondria) dysfunction has the potential to cause
widespread
damage to cells and tissues.
A number of diseases and disorders are thought to be caused by or be
associated with alterations in mitochondria) 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,
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
78
including but not limited to congenital muscular dystrophy with mitochondria)
structural abnormalities, fatal infantile myopathy with severe mtDNA depletion
and
benign "later-onset" myopathy with moderate reduction in mtDNA, MELAS
(mitochondria) encephalopathy, lactic acidosis, and stroke) and MIDD
(mitochondria)
diabetes and deafness); MERFF (myoclonic epilepsy ragged red fiber syndrome);
arthritis; NARP (Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE (Myopathy
and
external ophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy), LHON
(Leber's Hereditary Optic Neuropathy), Kearns-Sayre disease; Pearson's
Syndrome;
PEO (Progressive External Ophthalmoplegia); Wolfram syndrome; DIDMOAD
(Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, Deafness); Leigh's
Syndrome;
dystonia; schizophrenia; and hyperproliferative disorders, such as cancer,
tumors and
psoriasis.
According to generally accepted theories of mitochondria) function,
proper ETC respiratory activity requires maintenance of an electrochemical
potential
(~Lf) in the inner mitochondria) 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
mitochondria)
activity may also result in a catastrophic mitochondria) collapse that has
been termed
"mitochondria) permeability transition" (MPT). In addition, mitochondria)
proteins
such as cytochrome c and "apoptosis inducing factor" may dissociate or be
released
from mitochondria due to MPT (or the action of mitochondria) 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 mitochondria) activity may alternatively or additionally result
in the generation of highly reactive free radicals that have the potential of
damaging
cells and tissues. These free radicals may include reactive oxygen species
(ROS) such
as superoxide, peroxynitrite and hydroxyl radicals, and potentially other
reactive species
that may be toxic to cells. For example, oxygen free radical induced lipid
peroxidation
is a well established pathogenetic mechanism in central nervous system (CNS)
injury
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
79
such as that found in a number of degenerative diseases, and in ischemia
(i.e., stroke).
Mitochondria) involvement in the apoptotic cascade has been identified, for
example
mitochondria) 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.
There are, moreover, at least two deleterious consequences of exposure
to reactive free radicals arising from mitochondria) dysfunction that
adversely impact
the mitochondria themselves. First, free radical mediated damage may
inactivate one or
more of the myriad proteins of the ETC. Second, free radical mediated damage
may
result in catastrophic mitochondria) collapse that has been termed "transition
permeability". According to generally accepted theories of mitochondria)
function,
proper ETC respiratory activity requires maintenance of an electrochemical
potential in
the inner mitochondria) membrane by a coupled chemiosmotic mechanism. Free
radical
oxidative activity may dissipate this membrane potential, thereby preventing
ATP
biosynthesis and/or triggering mitochondria) events in the apoptotic cascade.
Therefore,
by modulating these and other effects of free radical oxidation on
mitochondria)
structure and function, the present invention provides compositions and
methods for
protecting mitochondria that are not provided by the mere determination of
free radical
induced lipid peroxidation.
For example, rapid mitochondria) permeability transition likely entails
changes in the inner mitochondria) 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 mitochondria) function characteristic of the mitochondria
associated diseases may also be related to loss of mitochondria) membrane
electrochemical potential by mechanisms other than free radical oxidation, and
such
transition permeability may result from direct or indirect effects of
mitochondria) genes,
gene products or related downstream mediator molecules and/or
extramitochondrial
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
genes, gene products or related downstream mediators, or from other known or
unknown causes. Loss of mitochondria) potential therefore may be a critical
event in
the progression of mitochondria associated or degenerative diseases.
Diabetes
5 Diabetes mellitus is a common, degenerative disease affecting 5 to 10
percent of the population in developed countries. The propensity for
developing
diabetes mellitus is reportedly maternally inherited, suggesting a
mitochondria) genetic
involvement (Alcolado et al., Br. Med. J. 302:1178-1180, 1991; Reny, Int. J.
Epidem.
23:886-890, 1994). Diabetes is a heterogenous disorder with a strong genetic
10 component; monozygotic twins are highly concordant and there is a high
incidence of
the disease among first degree relatives of affected individuals.
At the cellular level, the degenerative phenotype that may be
characteristic of late onset diabetes mellitus includes indicators of altered
mitochondria)
respiratory function, for example impaired insulin secretion, decreased ATP
synthesis
15 and increased levels of reactive oxygen species. Studies have shown that
diabetes
mellitus may be preceded by or associated with certain related disorders. For
example,
it is estimated that forty million individuals in the U.S. suffer from late
onset impaired
glucose tolerance (IGT). IGT patients fail to respond to glucose with
increased insulin
secretion. A small percentage of IGT individuals (5-10%) progress to insulin
deficient
20 non-insulin dependent diabetes (NIDDM) each year. Some of these individuals
further
progress to insulin dependent diabetes mellitus (IDDM). These forms of
diabetes
mellitus, NIDDM and IDDM, are associated with decreased release of insulin by
pancreatic beta cells and/or a decreased end-organ response to insulin. Other
symptoms
of diabetes mellitus and conditions that precede or are associated with
diabetes mellitus
25 include obesity, vascular pathologies, peripheral and sensory neuropathies,
blindness
and deafness.
Due to the strong genetic component of diabetes mellitus, the nuclear
genome has been the main focus of the search for causative genetic mutations.
However, despite intense effort, nuclear genes that segregate with diabetes
mellitus are
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
81
known only for rare mutations in the insulin gene, the insulin receptor gene,
the
adenosine deaminase gene and the glucokinase gene. Accordingly, mitochondria)
defects, which may include but need not be limited to defects related to the
discrete
non-nuclear mitochondria) genome that resides in mitochondria) DNA, may
contribute
significantly to the pathogenesis of diabetes mellitus (Anderson, Drug Dev.
Res. =16:67-
79, 1999).
A number of mitochondria) mutations associated with diabetic
phenotypes have been described (for reviews, see Gerbitz et al., Biochim.
Biophys. Acta
1271:253-260, 1995, or Rotig et al., Diabetes Metab. 22:291-298, 1996). A
number of
such mutations occur in genes encoding factors involved in protein translation
within
mitochondria, such as mitochondria) tRNAs (see, e.g., Suzuki et al., Diabetes
Care
17:1428-1432, 1994; Kishimoto et al., Diabetologia 38:193-200, 1995; van der
Ouweland et al., Muscle Nerve Suppl. 3: S 124-S 130, 1995; Hanna et al., Am.
J. Hum.
Genet. X6:1026-1033, 1995; Sano et al., J. Neurol. 243:441-444, 1996; Kameoka
et al.,
Biochenz Biophys. Res. Commun. 24:523-527, 1998; and Hirai et al., J. Clin.
Endocrinol. Metab. 83:992-994, 1998). Because mitochondria) translation is
dependent
on 4yr (Cote et al., J. Biol. Chem. 26:8487-8490, 1989; Cote et al., J. Biol.
Chem.
265:7532-7538, 1990), alterations in Dyr 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 Dye of the invention.
Furthermore,
agents that increase and/or stabilize 0~ 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 OW of the invention can also be used to estimate which agents)
are most
likely to be effective for a given individual, in that a patient having
mitochondria that
exhibit an altered Dyr is expected to be more likely to respond to agents that
modulate
Dy than a patient having mitochondria with a normal 4y.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
82
Parkinson's Disease
Parkinson's disease (PD) is a progressive, chronic, mitochondria
associated neurodegenerative disorder characterized by the loss and/or atrophy
of
dopamine-containing neurons in the pans 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.
It has been shown that the neurotoxin 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) induces parkinsonism in animals and man at least in
part
through its effects on mitochondria. MPTP is converted to its active
metabolite, MPP+,
in dopamine neurons; it then becomes concentrated in the mitochondria. The
MPP+
then selectively inhibits the mitochondria) enzyme NADH:ubiquinone
oxidoreductase
("Complex I"), leading to the increased production of free radicals, reduced
production
of adenosine triphosphate, and ultimately, the death of affected dopamine
neurons.
Apoptotic cell death is thought to constitute the terminal process in some
neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. It
has been
proposed that agents that help to maintain Dy might offer novel agents for
preventing or
treating neurodegenerative apoptosis (Tatton et al., Ann. Neurol. 4-1:5134-
5141, 1998).
Individuals suspected of having or being predisposed to developing Parkinson's
disease
(PD) may be identified using the ET-based assay ~yJ of the invention.
Moreover, the
ET-based Dy assay of the invention can be used to identify and characterize
compounds
that enhance or stabilize Dy, 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 Ayr of the invention can also be used to estimate which
agents) are
most likely to be effective for a given individual, in that a PD patient
having
mitochondria that exhibit an altered Dyr is expected to be more likely to
respond to
agents that modulate Dyr than a PD patient having mitochondria with a normal
Dy.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
83
Alzheimer's Disease
Alzheimer's disease (AD) is a chronic, progressive neurodegenerative
disorder that is characterized by loss and/or atrophy of neurons in discrete
regions of the
brain, and that is accompanied by extracellular deposits of ~3-amyloid and the
intracellular accumulation of neurofibrillary tangles. It is a uniquely human
disease,
affecting over 13 million people worldwide. It is also a uniquely tragic
disease. Many
individuals who have lived normal, productive lives are slowly stricken with
AD as
they grow older, and the disease gradually robs them of their memory and other
mental
faculties. Eventually, they cease to recognize family and loved ones, and they
often
I 0 require continuous care until their eventual death.
Mitochondria) 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 mitochondria) physiology may be among the earliest events in PCD
(Zamzami
et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Mecl 181:1661-
72, 1995)
and elevated reactive oxygen species (ROS) levels that result from such
altered
mitochondria) function may initiate the apoptotic cascade (Ausserer et al.,
Mol. Cell.
Biol. I x:5032-42, 1994). Indeed, one hallmark pathology of AD is the death of
selected
neuronal populations in discrete regions of the brain. Cell death in AD is
presumed to
be apoptotic because signs of programmed cell death (PCD) are seen and
indicators of
active gliosis and necrosis are not found (Smale et al., Exp. Neurolog.
133:225-230,
1995; Cotman et al., Molec. Neurobiol. 10:19-45, 1995.) The consequences of
cell
death in AD, neuronal and synaptic loss, are closely associated with the
clinical
diagnosis of AD and are highly correlated with the degree of dementia in AD
(DeKosky
et al., Ann. Neurology 2757-464, 1990).
In several cell types, including neurons, reduction in the mitochondria)
membrane potential (~~I') precedes the nuclear DNA degradation that
accompanies
apoptosis. In cell-free systems, mitochondria), 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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
84
resting mitochondria) membrane potentials than the corresponding parental SH-
SYSY
cell line, and cyclosporin A reverses the depressed OW in the AD cybrids
(Cassarino et
al., Biochem. Biophys. Res. Commun. 248:168-173, 1998). Individuals suspected
of
having or being predisposed to developing AD may be identified using the ET-
based
assay 4y~ of the invention. Moreover, the ET-based Dyr assay of the invention
can be
used to identify and characterize compounds that enhance or stabilize OW, 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 Dyr of the
invention
can also be used to estimate which agents) are most likely to be effective for
a given
individual, in that an AD patient having mitochondria that exhibit an altered
O~r is
expected to be more likely to respond to agents that modulate ~yJ than an AD
patient
having mitochondria with a normal Dy.
Other Neurological Disorders
Similar theories have been advanced for analogous relationships between
mitochondria) defects and other neurological diseases, including Alzheimer's
disease,
Leber's hereditary optic neuropathy, schizophrenia, "mitochondria)
encephalopathy,
lactic acidosis, and stroke" (MELAS), and "myoclonic epilepsy ragged red fiber
syndrome" (MERRF).
Increasing evidence points to the fundamental role of mitochondria)
dysfunction in chronic neurodegenerative diseases (Beal, Biochim. Biophys.
Acta 1366:
211-223, 1998), and recent studies implicate mitochondria for regulating the
events that
lead to necrotic and apoptotic cell death (Susin et al., Biochim. Biophys.
Acta 1366:
151-168, 1998). Stressed (by, e.g., free radicals, high intracellular calcium,
loss of
ATP, among others) mitochondria may release pre-formed soluble factors that
can
initiate apoptosis through an interaction with apoptosomes (Marchetti et al.,
Cancer
Res. 56:2033-2038, 1996; Li et al., Cell 91:479-489, 1997). Release of
preformed
soluble factors by stressed mitochondria, like cytochrome c, may occur as a
consequence of a number of events. In any event, it is thought that the
magnitude of
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
stress (ROS, intracellular calcium levels, etc.) influences the changes in
mitochondria)
physiology that ultimately determine whether cell death occurs via a necrotic
or
apoptotic pathway. To the extent that apoptotic cell death is a prominent
feature of
degenerative diseases, mitochondria) dysfunction may be a critical factor in
disease
5 progression. To the extent that 4y 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
Ayr of the invention. The ET-based Dye assay of the invention can also be used
to
identify and characterize agents that enhance or stabilize Dy, and these
agents are
10 expected to have remedial, therapeutic, palliative, rehabilitative,
preventative,
prophylactic or disease-impeditive effects on patients suffering from, or
thought to be
predisposed to developing, such disorders. The ET-based assay of Dy of the
invention
can also be used to estimate which agents) are most likely to be effective for
a given
individual, in that a patient having mitochondria that exhibit an altered 4~r
is expected
15 to be more likely to respond to agents that modulate Dyl than a patient
having
mitochondria with a normal Dyr.
Stroke
In contrast to chronic neurodegenerative diseases, neuronal death
following stroke occurs in an acute manner. A vast amount of literature now
documents
20 the importance of mitochondria) function in neuronal death following
ischemia/reperfusion injury that accompanies stroke, cardiac arrest and
traumatic injury
to the brain. Experimental support continues to accumulate for a central role
of
defective energy metabolism, alteration in mitochondria) function leading to
increased
oxygen radical production and impaired intracellular calcium homeostasis, and
active
25 mitochondria) participation in the apoptotic cascade in the pathogenesis of
acute
neurodegeneration.
A stroke occurs when a region of the brain loses perfusion and neurons
die acutely or in a delayed manner as a result of this sudden ischemic event.
Upon
cessation of the blood supply to the brain, tissue ATP concentration drops to
negligible
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
86
levels within minutes. At the core of the infarct, lack of mitochondria) 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
mitochondria) ATP. Cell death in acute neuronal injury radiates from the
center of an
infarct where neurons die primarily by necrosis to the penumbra where neurons
undergo
apoptosis to the periphery where the tissue is still undamaged (Martin et al.,
Brain Res.
Bull. 46:281-309, 1998).
Much of the injury to neurons in the penumbra is caused by
excitotoxicity induced by glutamate released during cell lysis at the infarct
focus,
especially when exacerbated by bioenergetic failure of the mitochondria from
oxygen
deprivation (MacManus and Linnik, J. Cerebral Blood Flow Metab. 17:815-832,
1997).
The initial trigger in excitotoxicity is the massive influx of Ca'+ primarily
through the
NMDA receptors, resulting in increased uptake of Ca'l into the mitochondria
(reviewed
by Dykens, "Free radicals and mitochondria) dysfunction in excitotoxicity and
neurodegenerative diseases" in Cell Death and Diseases of the Nervozrs System,
V. E.
Koliatos and R.R. Ratan, eds., Humana Press, New Jersey, pages 45-68, 1999).
The
Ca' ~ overload collapses the mitochondria) membrane potential (OBI'",) and
induces
increased production of reactive oxygen species (Dykens, J Neurochem 63:584-
591,
1994; Dykens, "Mitochondria) radical production and mechanisms of oxidative
excitotoxicity" in The Oxygen Paradox, K.J.A. Davies, and F. Ursini, eds.,
Cleup Press,
U. of Padova, pages 453-467, 1995). If severe enough, O~Ym collapse and
mitochondria)
CaZ+ sequestration can induce opening of a pore in the inner mitochondria)
membrane
through a process called mitochondria) 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 Acta 1241:139-176,
1995;
Ellerby et al., J Neurosci 17:6165-6178, 1997). Consistent with these
observations,
glutamate-induced excitotoxicity can be inhibited by preventing mitochondria)
Ca2+
uptake or blocking MPT (Budd et al., J. Neurochem 66:403-411, 1996; White et
al., J.
Neurosci 16:5688-5697, 1996; Li et al., Brain Res 753:133-140, 1997; Stout et
al., Nat.
Neurosci. 1:366-373, 1998).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
87
Agents and methods that maintain mitochondria) integrity during
transient ischemia and the ensuing wave of excitotoxicity would be expected to
be
novel neuroprotective agents with utility in limiting stroke-related neuronal
injury.
Given the limited therapeutic window for blockade of necrotic death at the
core of an
infarct, it may be particularly desirable to develop therapeutic strategies to
limit
neuronal death by preventing mitochondria) dysfunction in the non-necrotic
regions of
an infarct. As explained in more detail in Example 9 herein, such agents may
be
isolated by screening collections of compounds for their ability to stabilize
Dy 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 Dye of the invention can also be used to
estimate
which agents) are most likely to be effective for a given individual, in that
a patient
having mitochondria that exhibit an altered O~J is expected to be more likely
to respond
to agents that modulate 4y than a patient having mitochondria with a normal
~y.
Hyperproliferative Disorders
Whereas mitochondria-mediated apoptosis may be critical in
degenerative diseases, it is thought that disorders such as cancer involve the
unregulated
and undesirable growth (hyperproliferation) of cells that have somehow escaped
a
mechanism that normally triggers apoptosis in such undesirable cells. Enhanced
expression of the anti-apoptotic protein Bcl-2 and its homologues is involved
in the
pathogenesis of numerous human cancers. Bcl-2 acts by inhibiting programmed
cell
death and overexpression of Bcl-2, and the related protein Bcl-XL, block
mitochondria)
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. Nat). Acad. Sci. U.S.A. 94:6939-6942, 1997). In contrast,
overexpression of
Bcl-2 and Bcl-XL protect against the mitochondria) dysfunction preceding
nuclear
apoptosis that is induced by chemotherapeutic agents. In addition, acquired
multi-drug
resistance to cytotoxic drugs is associated with inhibition of cytochrome c
release that is
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
88
dependent on overexpression of Bcl-XL (Kojima et al., J. Biol. Chem. 273:
16647-
16650, 1998).
There is a need for compounds and methods that inhibit the growth or
enhance the death of cells and tissues that have escaped appropriate apoptotic
signals, as
well as cytotoxic agents that cause the death of undesirable (e.g., cancer)
cells by
triggering the apoptotic cascade or otherwise. In particular, because
mitochondria are
mediators of apoptotic events, agents that stimulate mitochondrially mediated
pro-
apoptotic events would be especially useful. Because mitochondria have been
implicated in apoptosis, it is expected that agents that interact with
mitochondria)
components will effect a cell's capacity to undergo apoptosis. Such agents are
expected
to have remedial, therapeutic, palliative, rehabilitative, preventative,
prophylactic or
disease-impeditive effects on patients suffering from, or thought to be
predisposed to
developing, hyperproliferative diseases such as cancer and psoriasis. The ET-
based
assay of Dye of the invention can also be used to estimate which agents) are
most likely
to be effective for a given individual, in that a patient having mitochondria
that exhibit
an altered Dye is expected to be more likely to respond to agents that
modulate Dyr than
a patient having mitochondria with a normal Dyr.
The ET-based assay of mitochondria) Dy of the invention may also be
used to identify agents that are selectively cytotoxic for hyperproliferative
or other
undesirable cell types. For example, Dye is elevated in some carcinoma cell
lines, and
agents that accumulate in mitochondria as a function of O~r (such as rhodamine
123) are
preferentially cytotoxic to such carcinoma cells (Modica-Napolitano et al.,
Cancer Res.
=17:4361-4365, 1987; Andrews et al., Cancer Res. 52:1895-1901, 1992).
In sum, the invention may be used to develop assays for subcellular
conditions or intracellular processes, such as changes in mitochondria) Ayr,
in order to
identify and characterize agents to treat degenerative disorders and diseases
as well as
hyperproliferative diseases. The ET-based assay of Dyr 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.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
89
The following examples illustrate the invention and are not intended to
limit the same. Those skilled in the art will recognize, or be able to
ascertain through
routine experimentation, numerous equivalents to the specific substances and
procedures described herein. Such equivalents are considered to be within the
scope of
the present invention.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
EXAMPLES
EXAMPLE 1
SELECTION OF COMPOUNDS AND OPTIMIZAT10N
OF CONDITIONS FOR ET-BASED ASSAYS
5 In order to develop an ET-based assay to detect conditions within a
subcellular compartment (such as an organelle or a membrane-bounded portion
thereof),
and monitor changes thereof, it is necessary to determine appropriate pairs of
donor and
acceptor compounds and useful concentrations thereof. Using the information
and
methods presented herein, one skilled in the art can readily determine donor
and
10 acceptor ~ compounds, and concentrations thereof, appropriate for a variety
of such
assays.
One step in the process of developing an ET-based assay involves
optimizing concentrations of the donor and acceptor compounds, as well as
other
conditions for the assay. In general, with regards to the concentrations of
the donor and
15 acceptor compounds, at least two criteria apply. First, the concentrations
of the donor
and acceptor compounds should be sufficient for energy transfer to occur.
Second, the
concentration of each compound should be low enough that (a) any non ET-based
signal from the compounds is negligible, so that the background signal in the
assay is
minimal, and (b) any undesirable effects on cellular physiology, including
cellular
20 toxicity, and/or effects on the subcellular compartment of interest, are
minimal. It
should be noted, however, that not every compound will have undesirable
effects on
cellular physiology.
In the case of a FRET-based assay of Ayr using NAO and TMRM, these
criteria are applied as follows. Because NAO is known to be toxic to certain
cells at
25 higher concentrations, for example at > 10 pM as reported by Maftah et al.
(FEBS Lett.
260:236-240, 1990), NAO sensitivity of cells to be used should first be
determined to
avoid exposing cells to toxic levels of this ET molecule. The NAO
concentration that is
toxic may vary depending on the cell (e.g., a cell line) selected for use in a
given
experiment, and on other conditions such as duration of exposure to NAO, the
presence
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
91
of other toxic or protective factors are present, and the like. The person
having ordinary
skill in the art will, however, be able to select and readily determine
suitable NAO
concentrations and/or durations of NAO exposure to cells without undue
experimentation, based on the present disclosure.
For instance, a series of experiments was performed to determine the
optimum ratio and concentrations of NAO and TMRM for a FRET-based assay of Dy.
These tests can be applied to other pairs of donor and acceptor compounds in
other ET-
based assays.
General Protocols
The FRET-based assay of Dy was generally carried out in the following
manner, although variations to these general procedures can be made without
affecting
the sensitivity, accuracy or efficiency of the assay.
Cell Lines and Preparation Thereof
A variety of cell lines were used in the following experiments. The
neuroblastoma SH-SYSY is a multiply subcloned cell line of human origin (Perez-
Polo
et al., Dev. Neurosci. 5:418-423, 1982). SH-SYSY is a well-characterized cell
line that
is capable of differentiating into neuron-like cells, and is an accepted
cellular model for
a variety of neuronal cell functions (for reviews, see, e.g., Vaughan et al.,
Gen.
Pharmacol. 26:1191-1201, 1995; Pahlman et al., Acta Physiol. Scand. Suppl.
592:25
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 incorporated by reference). The 1685
cybrid cell
line is one example of a cybrid cell line of this type. The 1685 cybrid cell
line was
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
92
created by fusing platelets from an AD donor with SH-SYSY neuroblastoma cells
that
had been made rho° by extended treatment with ethidium bromide.
"MixCon"
designates a Mixed Control composed of cybrids prepared in like fashion but
using
platelets from n normal age-matched patients (n = 2-3, depending on the
particular
experiment) for the construction of the cybrid cells.
NCI-H460 is a human lung large cell carcinoma cell line available from
the American Type Culture Collection (ATCC, Manassas, VA) under accession No.
ATCC HTB-177. A preferred cellular medium for NCI-H460 cells is 90% (RPMI 160
medium with 2 mM L-glutamine, 1.5 g/L sodium carbonate, 4.5 g/L glucose, 10 mM
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 mitochondria) 0~ 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).
In general, cells were plated at about 2 to 3 x 104 cells per well on 96-
well microplates (CostarTM, black wall; clear, flat bottom) at least about 24
hours prior
to the assay. HBSS was generally used as cellul medium, but any media
appropriate for
a given cell line may be used in the assay.
Preparation of Donor and Acceptor Compounds
A 5 mg/ml stock solution of the ET donor compound, nonylacridine
orange (NAO, Molecular Probes, Inc., Eugene, OR; catalog #A1372), was prepared
in
DMSO. The stock solution was aliquoted into microfuge tubes and stored frozen
at
-20°C until thawed on ice immediately prior to the assay. Unless
otherwise specified,
for use in assays the 5 mg/ml NAO stock solution was diluted 1:5000 in Hank's
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
93
balanced salt solution (HBSS, Life Technologies, Grand Island, NY) to yield a
working
stock solution containing 1 pg/ml NAO, which was further diluted as indicated
below.
A stock solution of acceptor compound, 100 mM TMRM (Molecular
Probes, Inc., Eugene, OR; catalog #T668), was prepared in DMSO. This
concentration
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.
Instrument Preparation
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.
In the FLIPRTM instrument, there are positions for three 96-well
microplates. 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. In a typical assay, the first reagent 96-well (8 rows, 12
columns) plate was
set up so that the wells in Row A contained media (typically, HBSS), the wells
in Row
B contained a Dyr collapsing agent (typically, CCCP), and the remaining Rows
(C
through H) contained the test compounds) (e.g., candidate agents).
Furthermore, in a
Type II assay (see Example 5), a second reagent plate was set up so that each
well
contained an appropriate amount of a Dyr collapsing agent (typically, CCCP) to
be
added to the samples sometime after the test compound(s).
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
94
Fluorophore Loading
Fifteen minutes prior to the assay, the entire plate was gently flicked
over a sink to remove the cell media. The displaced media was replaced in each
well
with 100 p,1 HBSS that contained 5 pM TMRM and was prewarmed to 37°C.
In
general, it was preferred to prewarm media and reagents to 37°C and to
maintain cells at
37°C in order to avoid thermal shock that can itself cause changes in
Dy or cause the
death of sensitive cells. Ten minutes later, 20 p1 of the NAO working stock
solution
prepared as described above (1 pg/ml NAO) was added to each well (final
concentration, 200 ng/ml, equal to 0.4 p,M).
In an optional step, after letting the cells incubate in the presence of both
fluorophores for about 5 minutes, excess fluorophore was removed by gently
flicking
the plate to remove cell media and adding 100 u1 prewarmed HBSS to each well;
this
process was repeated up to three times. After the final plate flicking, 100
p,1 prewarmed
HBSS was added to each well. Depending on cell type used in a particular
experiment,
the cells could be incubated for varying periods of time prior to addition of
the test
compounds) (e.g., candidate agents), with no appreciable loss of ET signal. In
the case
of SH-SYSY cells, this incubation period was up to approximately 40 minutes.
Assay Readings
Prior to the addition of test compounds, about 20 readings were taken on
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 Dye. For example, if
cellular
integrity was compromised, a significant collapse in Dyr 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.
To determine the ET value corresponding to maximal collapse of Ayr
(i.e., Dyr ~ 0 in theory), a Dy collapsing agent (e.g., CCCP) was added as
follows. In
Type I assays (Figure 3A), the collapsing agent was added to wells distinct
from those
receiving the test compounds at roughly the same time that the test compounds
were
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
added, and readings of these wells were taken at the same time as readings
were taken
of the wells that received test compounds. In Type II assays (Figure 3B), the
collapsing
agent was added to the same wells that received the test compounds after
readings had
been taken at 5-second intervals for a period of time (typically about 9-1 S
minutes), and
5 readings were then taken for a second period of time roughly equivalent to
the first
period of time.
Data Analysis
The assay results are presented as plots of relative fluorescence units
(RFU) over time (Figure 6) for qualitative analysis. For quantitative
analyses,
10 calculations were as follows:
For Type I assays, the initial instrument reading for each well was set to
zero. The readings taken at S-second intervals following those taken at 3-
second
intervals to verify cellular integrity, typically readings numbered from about
reading 21
to about reading 195-200, were summed (EF~). Tests of significance for
multiple (i.e.,
15 >2) groups, such as one-way ANOVA of treatment groups with no transform,
Newman-
Keuls or Bonferroni (Dune's) multi-comparisons, were used to evaluate the
significance
of results.
For Type II assays, the initial instrument reading for each well was set to
zero, and readings taken at 5-second intervals (following integrity
confirmation as
20 described above) numbered from about 21 to about 195-200 were summed
(EF,~). For
normalization, the readings during the final 4 minutes (i. e., readings
numbers about 214
to 230) after addition of the O~r collapsing agent (CCCP) to maximally
compromise
membrane potential were averaged (F~ccP). Because the use of ratios would
violate
mathematical assumptions inherent in ANOVA algorithms, the data were
transformed
25 (log or arcsin) before being evaluated for significance in one-way ANOVA
analyses.
For either type of assay, sums and averages for each well were calculated
using the software provided with the FLIPRTM instrument, exported into EXCELTM
(Microsoft, Inc., Redmond, WA) via *.txt, and finally exported into GB Stat
(Dynamic
Microsystems, Silver Springs, MD) for ANOVA. FLIPRTM kinetic data are exported
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
96
into EXCEL for mean and standard error calculations of the readings taken over
the
time courses. It was desirable (but not necessary) to back up all FLIPRTM data
on CD,
or another appropriate machine-readable format, on a daily basis.
Results
In an initial set of experiments, MixCon cybrid cells were treated with
six different concentrations of TMRM (0, 1.25, 2.5, 5.0, 10 and 20 ~M) and NAO
(0, 6,
12, 25, 50, 100, 200 and 400 ng/ml; respectively, 0, 13, 26, 52, 105, 210, 420
and 840
nM) on a 96-well plate. Each of the 48 possible combinations of TMRM and NAO
concentrations was tested in duplicate using a FLIPR instrument using a 0.1
second
shutter with the laser set at 300 mW and readings taken using a 510-590 nm
filter.
CCCP was added to all samples ( 1.5 ~,M) at 1 minute after the plate was put
into the
FLIPRTM instrument.
According to non-limiting theory, if FRET occurred between NAO and
TMRM, which localize to the inner mitochondrial membrane and the mitochondrial
matrix, respectively, then a change in FRET-based signal should occur
following CCCP
addition. Thus, the addition of CCCP would cause Dy to be decreased and, as a
consequence, the mitochondrial concentration of the acceptor compound (TMRM)
would also decrease as TMRM exited the mitochondria and/or was taken up to a
lesser
degree by mitochondria. Because the donor compound (NAO) is retained by
mitochondria regardless of Dyr, the donor and acceptor compounds would cease
to be in
sufficient proximity to one another for FRET to occur, and the signal
resulting from
FRET should decrease, as indicated by a change in fluorescence (expressed in
relative
fluorescence units, RFU).
Also according to non-limiting theory, the energy transfer from NAO to
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
97
emission wavelength [~,A(em)]. 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., 7~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).
In the present example, FRET was measured indirectly. TMRM + NAO
loaded SYSY 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 (i. e., increasing fluorescence at or near 7~Dem). In contrast, if
FRET had
been measured directly, the signal at or near 7~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.
The results of indirect FRET measurement are shown in Figure 4. In
these results, FRET was seen as an increase in signal (dequenching of NAO
emission)
that occurred following CCCP addition only when both donor and acceptor
compounds
were present at a given set of concentrations, i. e., the increase did not
occur when either
the acceptor or donor compound alone was present at the same concentration.
For
example, in Figure 4, FRET occurred in wells E9, E10, F9, F10, 9G and IOG, as
contrasted with the signals in wells A9 and A10 (NAO absent) and wells F1 and
F2
(TMRM absent). Although FRET was probably occurring in other wells in the
extreme
upper right-hand portion of Figure 4, the signal in these wells may also
include a
significant background signal component derived from NAO alone (e.g., compare
wells
H 11 and H 12 to H 1 and H2) or TMRM alone (e. g., compare wells H 11 and H 12
to A 11
and A12). Based on these results, useful preferred concentrations of NAO and
TMRM
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
98
for the assay include 50 ng/ml NAO and 10 ~M TMRM (wells E9 and E 10), 100
ng/ml
NAO and 10 ~M TMRM (wells F9 and F 10), 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).
The data in Figure 4 were analyzed as described above for the Type I
assay, i. e., the initial reading for each well was set to zero, and the RFU
readings taken
from about 21 to about 175 seconds were summed (EF,~). The results for varying
concentrations of TMRM were graphed as a function of NAO concentration, as
shown
in Figure 5. FRET occurred at 50, 100 and 200 ng/ml of NAO with 5 or 10 ~M
TMRM, as evidenced by the increase in signal at these concentrations (e.g.,
compare the
5 and 10 ~M TMRM curves in the 50-200 ng/ml NAO range with the 0, 1.25 and 2.5
~M TMRM curves in the same range of NAO concentrations).
In another experiment designed to examine the background signal from
each fluorophore individually as well as time course of CCCP-mediated Dye
collapse,
FRET was measured in cells treated with either 5 ftM TMRM , 420 nM NAO, or
with
both compounds, for a more extended period after CCCP addition (l.~ ~M). As
shown
in Figure 6, a rapid increase in fluorescence occurred within the first two
minutes after
CCCP addition, after which the change in fluorescence reached a plateau. When
either
NAO or TMRM was present alone, the fluorescent signal was essentially
constant.
In order to determine if the NAO dequenching signal that was measured
in the FRET-based assay might be linear over different cell densities, the
following
experiments were performed. Different numbers of MixCon or 1685 cybrid cells
were
preincubated in replicate in wells of 96-well plates for about 10 minutes with
5 ~M
TMRM, after which 4 ng/ml NAO was added and the cells were incubated for an
additional 5 minutes. Finally, CCCP ( 1 ~,M) was added to each well, and
fluorescence
signals were monitored at 530 + 25 nm using a FLIPRTM device. Mitochondrial
efflux
of TMRM then took place, as evidenced by an increase in fluorescence signal
corresponding to the dequenching of NAO emissions over time. The initial
slopes of
the curves (RFU over time) were plotted against the number of cells per well.
The
results showed that the Dyr-dependent fluorescent signal increased in a linear
fashion
over the range of from about 38,000 to about 330,000 cells per well.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
99
Although many of the experiments described herein made use of a
FLIPR instrument, and involved a series of measurements over time, the
invention may
be carried out using any instrument or device of sufficient sensitivity and
capable of
monitoring at least two time points (i. e., before and after addition of an
agent that
affects Dyr). In one experiment, for example, MixCon and 1685 cybrid cells
were
preincubated with TMRM and NAO as above, and fluorescence at 538 nm was
measured using an fmaxT"" (Molecular Devices, Inc., Sunnyvale, CA)
fluorimetric plate
reader (excitation = 485 nm) and then treated with CCCP (final concentration,
1.3 pM).
Ten minutes later, the fluorescence at 538 nm was again determined, and found
to have
increased significantly as compared to control cells treated with buffer
alone, in all three
cell types (SYSY cells, MixCon cybrids, 1685 cybrids).
Moreover, the 1685 cybrid cell line, which comprises mitochondria from
a patient having Alzheimer's disease, was more sensitive to ionomycin, i. e.,
showed a
greater degree of loss of Dyr than the control cybrid cells (MixCon) or the
parental SH-
SYSY cell line. This result demonstrates that the assay can be used to detect
differences
among cell types in reactions to agents that influence Dy.
EXAMPLE 2
PARAMETER-DEPENDENT CO-LOCALIZATION
OF ACCEPTOR-DONOR COMPOUNDS
Another step in the process of developing an ET-based assay to detect
conditions within a subcellular compartment (such as an organelle or a
membrane-
bounded portion thereof), and monitor changes thereof, is to confirm that not
only do
the donor and acceptor compounds co-localize in sufficient proximity for
energy
transfer to occur, but also that such co-localization is dependent on the
state of the
parameter to be measured. That is, at least one of the compounds must localize
to
(accumulate in) the subcellular compartment of interest as a function of the
measured
parameter, and must leave that compartment and/or accumulate less rapidly or
efficiently in that compartment as that parameter changes.
For example, for an ET-based assay designed to measure Dyr of
mitochondria, one of the compounds (either the donor or the acceptor) must
accumulate
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
100
in and/or be released from mitochondria in a manner that is dependent on Dye,
whereas
the presence of the other compound (the acceptor or donor, respectively) in
mitochondria must be 4yr-independent. Combining these criteria with the
information
presented herein, one skilled in the art can readily choose donor-acceptor
combinations
that are appropriate for ET-based Dye assays.
Compounds whose mitochondria) concentration is not dependent on Dyr
include, by way of example and not limitation, NAO (Pent et al., Eur. J.
Biochem.
194:389-397, 1990; Maftah et al., Biochem. Biophys. Res. Comm. 164:185-190,
1989),
MitoTracker~ Green FM (U.S. Patent Nos. 5,459,268 and 5,686,261 ), MitoFluorTM
Green (Haugland, Handbook of Fluorescent Probes and Research Chemicals', 6th
Ed.,
Spence, ed., Molecular Probes, inc., Eugene, Oregon, 1996, page 269) and
fusion
proteins comprising (a) a red- or yellow-shifted Green Fluorescent Protein
polypeptide,
or a "FLASH" polypeptide, and (b) a polypeptide sequence that localizes the
fusion
protein to the mitochondria) matrix or inner membrane. These compounds are
listed as
Group IV and V donor compounds in Table 2. A series of representative Group IV
and
V acceptor compounds is also presented in Table 2. Of the Group IV and V
acceptor
compounds in Table 2, those that accumulate in mitochondria in a Dyr-dependent
manner include, by way of example and not limitation, rhodamine 123 (Emaus et
al.,
Biochim. Biophys. Acta 850:436-448, 1986; Scaduto et al., Biophys. J. 76:469-
477,
1999), TMRM and TMRE (Farkas et al., Biophys. J. 56:1053-1069, 1989; Ehrenberg
et
al., Biophys. J. X3:785-794, 1988).
With regard to specific sites of accumulation of these compounds, NAO
specifically interacts with the inner mitochondria) membrane (Maftah et al.,
Biochem.
Biophys. Res. Comm. 164:185-190, 1989). Without wishing to be bound by theory,
TMRM, TMRE and rhodamine 123 are believed to localize to the mitochondria)
matrix,
although a recent report indicates that these compounds additionally
accumulate
reversibly in the inner and outer aspects of the inner mitochondria) membrane,
possibly
as a function of localized microenvironments there featuring intensified
membrane
potential (Scaduto et al., Biophys. J. 76:469-477, 1999). Accordingly,
irrespective of
whether TMRM, TMRE and rhodamine 123 localize to the inner mitochondria)
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
101
membrane or the mitochondrial matrix (or both), they are expected to be in
close
proximity to the inner mitochondrial membrane, where NAO localizes (see Figure
1 ).
In order to confirm that FRET only occurs between appropriately
localized donor-acceptor pairs of compounds in living cells, the following
experiment
was carried out. SH-SYSY cells were cultured and assayed as in Example 1 with
the
following exceptions. Cells were incubated with an "acceptor" compound at 5 qM
for
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 Dyr, CCCP, was
added to
the cells at a concentration of 1 ~M, and relative fluorescence was measured
using an
10 fmaxT"" (Molecular Devices, Inc., Sunnyvale, CA) fluorimetric plate reader
(excitation,
485 nm; emission read at 538 nm + 20 nm). The mean rate of change in relative
fluorescent units (RFU) in 6 to 8 replicate wells was calculated as the slope
of the curve
over the initial 3.5 minutes using the software provided with the fmaxT""
instrument via
least squares linear regression.
The results are shown in Table 4. FRET was detected between NAO and
TMRM, which localize to the inner mitochondrial membrane and the mitochondrial
matrix, as indicated by the mean rate of RFU change following CCCP addition.
FRET
occurred between NAO and TMRM until the addition of CCCP, which caused a
decrease in Ayr and exit of the acceptor compound (TMRM) from mitochondria.
Because the donor compound (NAO) is retained by mitochondria independent of
Dyr,
the donor and acceptor compounds ceased to be in sufficient proximity to one
another
for FRET to occur, and the signal resulting from FRET declined (as indicated
by the
relatively rapid rate of change in RFU).
In contrast to the effect seen with NAO and TMRM, when calcein or
CO-Fluor were used as "donor" compounds, the rate of RFU change following CCCP
addition was negligible. This reflects the fact that, although calcein and CO-
Fluor have
emission peaks similar to that of NAO, they did not localize to mitochondria
and thus
were not in close enough proximity to the "acceptor" compound (the
mitochondrially
localized TMRM) for FRET to occur. In like fashion, when SNAFL, which does not
localize to mitochondria, was used as an "acceptor" compound and NAO was used
as a
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
102
"donor" compound, FRET was not observed, even though the excitation peak
wavelength of SNAFL (514 nm) is closer to emission peak wavelength of NAO (517
nm) than the excitation peak wavelength of TMRM (544 nm). Thus, as expected,
for
energy transfer to occur, both spectral overlap and physical proximity were
required.
Table 4: FRET Only Occurs Between Appropriately
Co-Localized Donor and Acceptor Compounds
"Donor" "Acceptor"
Compound Compound
Mean Rate
7~D(ex)~,D(em) ~,A(ex) ~,A(em) of RFU
Change
NAO 495 519 nm 548 nm 573 nm TMRM 0.3750
nm ***
Calcein 494 517 nm 548 nm 573 nm TMRM 0.0050
nm ***
CO-Fluor 492 517 nm 548 nm 573 nm TMRM 0.0025
nm ***
NAO 495 519 nm 514 nm 546 nm SNAFL 0.0025
nm ***
In sum, energy transfer (in this example, FRET) occurred only when the
ET donor and acceptor molecules were appropriately co-localized within the
subcellular
compartment of interest. Moreover, processes that caused an ET donor or ET
acceptor
molecule to localize to a different site in such a manner that the pair of ET
molecules
were no longer in sufficient proximity for energy transfer to occur were
monitored and
assayed by measuring changes in a signal generated as a result of the energy
transfer.
EXAMPLE 3
PARAMETER-DEPENDENT CHANGES IN ENERGY TRANSFER
The preceding Examples show how to determine energy transfer between
an ET donor and an ET acceptor molecule, how to optimize ET assay conditions
including concentrations of the donor and acceptor compound, and how to
demonstrate
that energy transfer is dependent upon co-localization of both compounds
within the
same or adjacent subcellular sites. In order to demonstrate that an ET-based
assay
detects the condition or parameter within a subcellular compartment of
interest, and
CA 02375542 2001-12-13
WO 00/79274 PCTNS00/17380
103
monitor changes thereof, it is useful to validate the assay with agents having
known
effects on the chosen condition or parameter.
Using a FRET-based assay designed to measure ~y~ of mitochondria as a
model, a variety of agents are known in the art to lower (dissipate) or
eliminate
(collapse) Ayr. Additionally, some agents are known to increase Dy above
normal
levels, i. e., to hyperpolarize mitochondria. Both types of agents were
evaluated using
the FRET-based assay of ~y~.
Agents that Increase Dyr
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. On the same 96-well plate, 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 Dyr
collapse, was
determined for eight separate wells of each of the three combinations of cells
and agents
using a FLIPRTM instrument.
If the agents work as expected, and according to non-limiting theory,
hyperpolarization should increase Dyr, leading to increased
intramitochondri~al TMRM
accumulation, leading in turn to increased energy transfer (i. e., NAO
quenching). The
results (Table 5) show that oligomycin had the predicted effect. That is,
because the
cells treated with oligomycin contained hyperpolarized mitochondria, the
initial FRET
signal was significantly less than that seen in cells that were not exposed to
oligomycin.
Table 5: Effect of Oligomycin on FRET-Based Assay of Dyr
Cells OligomycinInitial Significance* RelativeStandard
FRET to Error
Signal MixCon, No Oligomycin
MixCon (none) 521.1 ___ 17
MixCon 10 uM 296.2 P < 10 8 13
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
104
* Calculated via two-tailed t-test
Agents that Decrease Dy and Protective Agents:
Ionomycin and Bon~krekic Acid
The effect on Dyr of the calcium ionophore ionomycin, which dissipates
and eventually collapses Dyr, alone or in combination with bongkrekic acid
(BKA), was
compared to the effects of the Dy-collapsing agent CCCP. Because BKA binds to
the
adenine nucleotide translocator, the activity of which is required for
mitochondrial
permeability transition (MPT), it was predicted that BKA would have an
ameriolating
effect on the Dy dissipation caused by ionomycin. SH-SYSY cells were treated
with
donor and acceptor compounds (respectively, NAO, 420 nm, and TMRM, 5 ~M)
according to the procedure described in Example l, 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 Dy and exodus from the
mitochondria of the acceptor compound, TMRM. Treatment with ionomycin (Fig. 7,
"I") ultimately yielded a more gradual change in fluorescence, as was expected
for an
agent known in the art to cause a slower dissipation in Dyr than CCCP. The
addition of
BKA to ionomycin-treated cells (Fig. 7, "I+BKA") moderated the effect of
ionomycin
effects and ultimately resulted in a fluorescence signal that was similar to
that seen
when HBSS media only (Fig. 7, "MO") was added to the cells.
Ionomycin and Ruthenium Red
Ruthenium red was confirmed to have a protective effect with regard to
the Dy-dissipating effects of ionomycin. Ionomycin is an ionophore that
increases the
level of cytosolic calcium; this leads to a dissipation of Ayr as mitochondria
take up
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
105
calcium from the cytosol. Ruthenium red blocks the activity of the
mitochondria)
calcium uniporter, thus inhibiting or blocking mitochondria) uptake of
calcium.
Ruthenium red would therefore be expected to counteract the effect of
ionomycin. SH-
SYSY 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 Ayr was essentially completely blocked by ruthenium red.
Ionomycin or MPP+ and Cyclos orp in A
In another related experiment, cyclosporin A was confirmed to have a
protective effect with regard to the Dye-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 ~tM). 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. The results (Figure 9) demonstrate that the FRET-based assay
yields data
that follow the expected patterns, i. e., the ionomycin-mediated dissipation
of D~r was
inhibited by cyclosporin A. In other experiments, the assay was used to
confirm that
cyclosporin A ( 10 ~M, added 10 minutes prior to addition of the Dy agent)
essentially
blocked the long-term (> 10 minutes after addition) dissipation and collapse
of Dyr
otherwise caused by 0.5 mM MPP+.
Atractyloside and Cyclos orp in A
The FRET assay described above and in the preceding examples was
also validated by the fact that it showed a dissipation of Dy in SH-SYSY cells
treated
with atractyloside (ATR, 5 mM) that peaked at about 6 minutes after ATR
addition. At
this concentration of ATR, Dy recovered after about 15 minutes, whereas CCCP
(1
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
106
~M) led to a more complete collapse of OW that was maintained for at least 1 S
minutes.
Pretreatment with cyclosporin A (5 pM, 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.
In sum, energy transfer (in this example, FRET) occurred in a manner
that accurately reflected changes in a parameter (in these examples, Ayr)
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 Dy). Moreover, the measured activities of agents known to increase
(e.g.,
oligomycin) or decrease (e.g., CCCP. ionomycin, MPP+, ATR) the chosen
parameter
(~yr) were in agreement with their predicted effects. The same was true for
protective
agents (BKA, ruthenium red, cyclosporin A) that are known to counteract, in
whole or
in part, the effects of parameter-changing agents. These results indicate that
the ET-
based assay may be used to screen for and evaluate previously uncharacterized
compounds for their effects on the chosen parameter (in this example, O~r) and
for their
ability to counteract the effects of known compounds on the parameter of
interest.
EXAMPLE 4
EVALUATION OF ASSAY RESULTS
The results presented in the preceding examples demonstrate the need to
evaluate ET-based assay results in a fashion that yields meaningful
conclusions. Using
the results presented in Figure 7 as an example, although the initial rates of
change in
RFU of the samples treated with CCCP, ionomycin or ionomycin and BKA were
similar from about 78 seconds to about 127 seconds, the readings for these
three
samples diverged thereafter and were markedly different at 460 seconds. There
are a
variety of ways to evaluate the results of an ET-based assay, as summarized in
Table 6,
for example, using the results shown in Figure 7.
One method for evaluating ET-based assays is to measure the time taken
in each sample to reach a defined RFU value, i. e., to determine a threshold
intercept
value for each sample. Such a determination will indirectly reflect the
initial slope of
the curves. As shown in Table 6, however, selection of an appropriate
threshold
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
107
intercept RFU value is critical in this method of evaluation. Selecting
RFU=2220, for
example, as the intercept yields results that are inconsistent with the
expected effects on
Dye of the various treatments (i.e., CCCP > ionomycin > ionomycin & BKA >
media
only). Moreover, the RFU=2220 results are also somewhat confounding as the
sample
treated with ionomycin and BKA intercepts RFU=2220 twice. On the other hand,
selecting a lower intercept value (RFU=160) yields results having the expected
order.
In the latter case, however, the protective effects of BKA might not be fully
appreciated,
as the result for ionomycin plus BKA (0.345) is only slightly different than
that for
ionomycin alone (0.400).
Another method of evaluation of ET-based assays is to directly
determine the initial slope of the curve for each sample. However, as the
results shown
in Figure 7 demonstrate, data from different samples can yield curves having
similar
initial slopes, even thought the overall shapes of the curves and their
endpoints are
distinct.
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 Dyr (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.
Table 6: Different Evaluations of the Results in Figure 7
Treatment Area Time222o Timel6o
(from Ratio Time Ratio Time Ratio
to to to
expected Area media (min.) media (min.) media
to to
most to Under only Reach only Reach RFU only
least RFU
effect Curve Sample = 2220 Sample = 160 * Sample
on * * * *
OtV)
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
108
CCCP 16.2 10.1 0.85 0.133 0.050 0.083
x 105
ionomycin 6,79 4.22 0.55 0.086 0.200 0.333
x 105
ionomycin I = 0.75 1 = 0.117
and BKA 2.37 1.47 2 = 1.85 2 = 0.7110.255 0.425
x 105
media only 1.61 1.00 6.40 1.000 0.600 1.000
x 105
* Measured as sum of all readings over 0 to 460 seconds.
** Measured from moment when all 4 curves were coincident (t= 1.1 min.).
EXAMPLE 5
FRET-BASED ASSAY OF ~yI
The preceding Example illustrates a potential limitation in the "Type I"
FRET-based assay of Dye, in which the effects of various agents on Dy were
compared
to the effects of an agent (CCCP) that collapses Dy (Figure 3A). In order to
yield more
meaningful results, the "Type II" assay was developed. In the Type II assay,
the
agents) being tested is first added to a sample and, after allowing the
agents) being
tested some time to exert their effects, a Dye collapsing agent is
subsequently added to
the same sample in order to drive Dy to zero, thus establishing a baseline
value for the
results.
Figure 3B shows a Type II assay. In one version of the Type II assay,
wherein a compound is being tested for its ability to dissipate ~y, 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 Dyr-dissipating compound is added at timepoint
"2." If
the candidate Ayr-dissipating compound has little or no effect on Dyr, a
signal like that
represented by the solid line ("C"') is expected, whereas a test compound that
dissipates
Dyr results in a signal like that represented by the dashed line ("C"). At
timepoint "3,"
an agent that completely collapses Dy (e.g., CCCP) is added, and a reading
("D") is
taken after the collapse of0yr is complete, in order to allow for
normalization of the
various samples for variations in cell density, and in efficiency of loading
of the ET
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
109
donor and acceptor compounds. The Dy-dissipating activity of the test compound
is
calculated as the Dy-Dissipating Value according to the formula:
Dyl-Dissipating Value = (C-B) / (D-B)
wherein a higher value for the ~y~-Dissipating Value indicates a greater Dy-
dissipating
ability of the candidate compound.
In another version of the Type II assay, wherein a compound is being
tested for its ability to inhibit or enhance the activity of an agent that
dissipates Dyr, the
symbols in Figure 3B are as follows. An optional initial reading ("A") that
can be
normalized to zero is first taken. The test compound is added at timepoint
"1," and a
baseline measurement ("B") is taken. The 4yr-dissipating agent (e.g.,
ionomycin,
atractyloside, etc.) is added at timepoint "2." If the test compound has
little or no effect
on the activity of the Dye-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 Dyr-dissipating agent results in a signal like that
represented by the solid
line ("C' "). At timepoint "3," an agent that completely collapses OW (e.g.,
CCCP) is
added, and a reading ("D") is taken after the collapse of0y~ is complete in
order to allow
for normalization for variations in cell density and efficiency of loading of
the donor
and acceptor compounds. The activity of the test compound is calculated as the
Efficacy Index according to the formula:
Efficacy Index = (C-B) / (D-B)
wherein a lower value for the Efficacy Index indicates a greater protective
effect of the
test compound.
Although CCCP and ionomycin are used in the following exemplary
experiments, other Dyr collapsing agents are known and can be used. Such ~y~
collapsing agents include, by way of example and not limitation, valinomycin,
A23187
and 4-Br-A23187.
It is desirable to establish a dose-response curve for whatever Dyr
collapsing agent is used, as conditions for the Type II assay are preferably
such that Ayr
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
110
collapses, and the measured signal reaches a plateau, in a rapid manner (i.
e., preferably
within 5 minutes after addition of the Dyr 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 Dyr
collapsing agent.
A dose-response curve for CCCP is shown in Figure 10. In the
experiments performed to generate the data in this figure, SH-SYSY 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. These data also suggest that 10 pM was a near
saturating concentration of CCCP to use, as the response to 10 ~M CCCP was
only
slightly greater than that seen when 5 ~M CCCP was applied (compare to the
change in
responses between 2.5 uM and 5 uM CCCP).
The results from a representative Type II FRET experiment are shown in
Figure 11, which shows relative fluorescence units + standard errors for
readings taken
at the indicated timepoints. In this experiment, SH-SYSY cells were contacted
with
NAO and TMRM according to the procedure of Example l, 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 Dyr-dissipating agent 4-bromo-A23187. About 6.5 minutes later, the Dyr-
collapsing
agent CCCP (final concentration, 5 ~M) was added to all the samples and the
fluorescence was read for an additional 7.5 minutes. As shown in Figure 11,
the cells
treated with 4-bromo-A23187 ("4-BR") exhibited a gradual loss of Dyr up until
the time
CCCP was added, at which point Dy ftirther decreased and ultimately collapsed.
As
also shown in Fig. 11, the cells treated with media ("MO") also showed a rapid
loss of
Ayr following CCCP addition and approached complete OW collapse, the MO and 4-
BR
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
111
curves becoming asymptotic after about 600 seconds and for the remainder of
the
experiment.
EXAMPLE 6
DOSE RESPONSE CURVES FOR X141-DISSIPATING AND OL~I PROTECTIVE AGENTS
Having established the basic parameters of the ET-based assay of Dye,
more exact experiments were carried out to demonstrate that the assay can be
used to
generate dose-response curves for both OW-dissipating and 4y-protective
agents. SH-
SYSY cells were used in these experiments. The calcium ionophore ionomycin was
used as a mock candidate agent compound being evaluated for its capacity to
cause
dissipation of ~w, and cyclosporin A was used as a mock ionomycin-protective
agent.
Cells were grown to specific cell density and transferred to 96-well
plates as described above. For both sets of experiments, TMRM and NAO were
added
at the concentrations and in the order and timing described in Example 1. For
the
experiments involving ionomycin alone, ionomycin was added at various
concentrations 10 minutes after addition of NAO. In the case of the
experiments
designed to quantify the ability of cyclosporin A to protect against the
effects of
ionomycin, cells were loaded for 10 minutes with TMRM and for 5 minutes with
NAO
as described above for fluorophore (ET donor and acceptor molecules) loading,
following which the cells were washed and exposed to various concentrations of
cyclosporin A for 15 minutes prior to initiation of instrument readings.
Readings
numbered 1-21 were recorded at 3-second intervals, and thereafter readings
numbered
22-196 were recorded at 5-second intervals. As shown in Figure 13, the sum of
the
fluorescence signal over each time interval was determined and plotted against
the
log~~o~ ionomycin concentration (M) to generate a cyclosporin A dose-response
curve.
The dose response curves for cells exposed to ionomycin in three
separate experiments (50,000 cells per well in each experiment) are shown in
Figure 12.
The data generated parallel curves when plotted, demonstrating the
reproducibility of
the assay in analyzing compounds have a negative impact on Dyr.
The dose response curves for cells pretreated with varying amounts of
cyclosporin A and then exposed to ionomycin in three separate experiments
(39,000
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
112
cells per well in each experiment) are shown in Figure 13. The data generated
similar
curves when plotted, demonstrating the reproducibility of the assay in
analyzing
compounds that protect mitochondria from agents that have a negative impact on
Dyr.
EXAMPLE 7
S FRET IN VARIOUS CELL TYPES
In the preceding examples, the FRET-based assay of Dyr was performed
on a neuroblastoma cell line (SH-SYSY), and on the MixCon and 1685 cybrid cell
lines
that are generated from p° SYSY cells. Although the control (MixCon)
and
Alzheimer's (1685) cybrids show the same general response to various agents
and
treatments that influence Dyr, some differences were detected by the FRET-
based assay.
In this example, MixCon or 1685 cells (about 50,000 cells per well) were
preincubated
with 420 nM NAO and 5 ~M TMRM according to the procedure of Example 1, after
which the calcium ionophore A23187 (0 to S ~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-SYSY 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-SYSY cells. Statistical analysis (ANOVA) demonstrates that the increased
susceptibility of the AD (1685) cybrid cells was significant. Thus, the ET-
based assay
of Dyr of the present invention can be used to characterize mitochondria)
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 mitochondria) function), the assay may be used to aid
in the
diagnosis of such diseases.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
113
EXAMPLE 8
ET-BASED ASSAYS FOR DETECTING SPECIFIC CELL TYPES IN A SAMPLE
Assays utilizing energy transfer can be used to detect specific cell types
in a biological sample. For example, rhodamine 123 (a Group II. III and IV
acceptor
compound; see Table 2) is taken up rapidly and retained for long periods
(greater than
24 hours) by a variety of human carcinoma cells after washing, even though it
is not
usually well retained by other cell types when they are washed (Nadakavukaren
et al.,
Cancer Res. =15:6093-6099, 1985; Summerhayes et al., Proc. Natl. Acad. U.S.A.
79:5292-5296, 1982; Christman et al., Gynecol. Oncol. 39:72-79, 1990).
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.
The following experiment was carried out in order to demonstrate that
certain cell types (in this Example, a human carcinoma cell line)
differentially take up
and retain particular ET donor and/or acceptor molecules as provided herein,
and
therefore have unique properties permitting such specific cell types to be
detected by an
ET-based assay of the present invention, thereby distinguishing such cell
types from
others that may be present. NCI-H460 is a human lung large cell carcinoma cell
line
(see Example 1 for details). NCI-H460 cells were added to 96-well plates
(about
50,000 cells per well). In a Type II ~yf assay TMRM (5 ~M) and NAO (420 nM)
were
added to the cells according to the procedure of Example 1. Ionomycin (50 ~M)
in
media was also added to one set (n = 24) of samples and media only was added
to a
control set of samples. The Dy collapsing agent CCCP (5 ~M) was added to all
the
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
114
samples about 9 minutes later. Fluorescence was measured using a FLIPRTM
instrument
during the experiment, as described above.
The results are shown in Figure I5. Although ionomycin ("I") caused a
large degree of Dyr dissipation, the carcinoma cells recovered relatively
rapidly by about
6 minutes after addition of ionomycin. This recovery was unlike that seen with
the
cybrid cell lines or the neuroblastoma SH-SYSY cell line used in the preceding
Examples, and suggested that the mitochondria in the carcinoma cell line take
up
TMRM more rapidly, either in general or at least after a challenge to Dyr,
than did
mitochondria from other cell types. It is particularly noteworthy that
differential
susceptibility to inducers of O~I' collapse, as shown here by differential
sensitivity to
ionomycin detected in the FRET assay of mitochondria) membrane potential, can
be
used to distinguish cell types: The ionomycin concentration used here for NCI-
H460
cells (50 ~M), a concentration from which these cells recovered, was ten times
the
ionomycin concentration to which SH-SYSY cells were sensitive, as indicated by
their
I S loss of mitochondria) membrane potential (Fig. 12). As described above,
Figure 12
depicts increased dequenching of NAO fluorescence at higher ionomycin
conditions
using SH-SYSY cells, indicative of greater mitochondria) membrane potential
collapse
at the higher ionomycin concentrations, which effected the loss of TMRM from
the
mitochondria) compartment.
EXAMPLE 9
METHODS FOR IDENTIFYING COMPOUNDS FOR TREATING STROKE
Mechanisms of cell death from ischemia and reperfusion involve both
necrosis and delayed apoptosis, with mitochondria) dysfunction as a common
antecedent to both. A number of events follow ischemia-induced loss of
mitochondria)
function, including decreased mitochondria) energy metabolism, increased
mitochondria) production of toxic reactive oxygen species (ROS) after
reperfusion, and
active mitochondria) initiation of apoptotic cascades in conditions where
energy
production can be restored.
Following a neuronal ischemic event, mitochondria) ATP production
halts due to the lack of oxygen. Although glycolytic ATP production can
continue
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
115
under anoxic conditions, glycolysis cannot meet the energy demands of neurons
due to
limited stores of glycolysis substrates in the brain. Still, lactate does
accumulate in
anoxic brain tissue, providing a measurable endpoint for biologic assays.
Because of
losses in aerobic competence, the tissue ATP concentration drops to negligible
levels
within minutes after cessation of oxygen flow to the brain.
Without adequate ATP, the ATP-dependent ion transporters fail, and the
loss of ion homeostasis results in osmotic lysis and necrosis of neurons at
the anoxic
core of the infarct. De-energization also involves the loss of ATP-dependent
transport
processes that sequester glutamate. Massive influx of Ca'+ and other ions
ensues from
activation of voltage-dependent and ligand-dependent ion channels (White et
al., J.
Neurosci. 15:1318-1328, 1995; Harrington et al., Neuron 16:219-228, 1996;
Schinder et
al., J. Neurosci. 16:6125-6133, 1996). Upon reperfusion, high levels of
cytosolic Ca2+
directly activate mitochondria) calcium uptake, preventing the establishment
of normal
mitochondria) function upon re-introduction of oxygen. Excessive Ca2+
accumulation
in the mitochondria can potentiate the production of oxygen-and carbon-
centered
radicals in neurons and lead to inactivation of mitochondria) electron
transfer system
(Dykens, J. Neurochem. 63:584-591, 1994; Reynolds et al, J. Neurosci. 15:3318-
3327,
1995; Dugan et al., J. Neurosci. 15:6377-6388, 1995, Bindokas et al., J.
Neurosci.
16:1324-1336, 1996).
Another consequence of mitochondria) Ca2+ uptake is the induction of
the membrane permeability transition (MPT), the opening of a nonspecific,
voltage-
sensitive, pore that dissipates 0'1m and allows solutes of <1,500 Daltons to
equilibrate
across the inner mitochondria) membrane (see reviews, Zoratti et al., Biochim.
Biophys.
Acta 1241:139-176, 1995; Bernardi et al., J. Bioenerg. Biomemb. 26:509-517,
1994).
High 0'fm that is normally generated by the electron transport chain in the
absence of
high Ca2+ or free radical-induced injury, is a potent deterrent to MPT pore
formation.
Agents that moderate MPT and OLfm collapse, such as Bcl-2 and cyclosporin A,
correspondingly moderate glutamate excitotoxicity both in vitro and in vivo
(Hoyt et al.,
Br. J. Pharmacol. 122:803-808, 1997; Niemninen et al., Neurosci. 75:993-997,
1996;
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
116
Ankarcrona et al., FEBS Lett. 394:321-324, 1996; Uchino et al., Acta Physiol.
Scand.
155:469-471, 1995; Li et al., Brain Res. 753:133-140, 1997).
Failure of cellular Ca'-+ efflux mechanisms and activation of
phospholipases and proteases appear as late-stage events after ischemia and
can lead to
widespread damage to membranes and proteins. Cells exposed to less severe
stress may
initiate an apoptotic cascade. In this case, mitochondria may be reversibly
damaged and
release sufficient levels of apoptogenic factors to induce death while
maintaining a
residual capacity to generate ATP (MacManus et al., J. Cerebral Blood Flow
Metab.
17:815-832, 1997). Therefore, healthy mitochondria play a bifunctional role in
preservation of neuronal viability in ischemialreperfusion 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
mitochondria) integrity during ischemia/reperfusion and thereby defending
against the
ensuing wave of excitotoxicity thus permits identification of novel
neuroprotective
agents having utility for preventing stroke-related neuronal injury.
Primary Screening Assays
Measurement of Oll'", provides a comprehensive indication of
mitochondria) function and integrity. Therefore, the primary screening assay
in stroke
drug discovery utilizes the ET-based assay of 0~' in whole cells in a high-
throughput
format. Agents and methods that maintain mitochondria) 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 mitochondria) dysfunction in the non-necrotic regions of an
infarct. To this
end, compounds are screened for their effects on OLl' under control and Ca2+
overload
conditions.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
117
Following a stroke, much of the injury to neurons in the penumbra is
caused by excitotoxicity induced by glutamate released during cell lysis at
the infarct
focus. In order to more closely mimic in vivo biochemical and cellular events,
primary
screening assays are carried out in cells comprising one or more types of
glutamate
receptors (for reviews, see Gasis et al., Curr. Opin. Neurobiol. 1:20-26,
1991;
Westbrook, Curr. Opin. Neurobiol. 4:337-346, 1994; and Lynch et al., Curr.
Opin.
Neurobiol. 7:510-516, 1994).
Glutamate receptors include ionotropic glutamate receptors (iGIuRs) 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, NMDARl/2A, NMDAR1/2B, NMDAR1!2C and NMDARI/2D. AMPA
receptors are homomeric complexes including, for example, GluRl, GluR2, GluR3
and
GluR4. Kainate receptors may be either homomeric or heteromeric complexes of
GIuRS, GluR6, GluR7, KA-1 and KA-2. The mGIuRs are 7-transmembrane G-protein
coupled receptors that are also classified further into subclasses. Some
mGIuRs are
phospholipase C-coupled mGIuRs that increase cytosolic calcium; these include
mGluRl and mGluRS. Other mGluRs are adenylate cyclase-coupled mGluRs that
decrease cytosolic cAMP; these include mGIuR2, mGluR3, mGluR4, mGluR7, and
mGluRB.
One example of a cell comprising one or more types of glutamate
receptors that are used in primary screens is a primary cortical neuron
expressing
endogenous NMDA receptors. In these cells, application of extracellular
glutamate
elevates intracellular calcium levels (Stout et al., Nat. Neurosci. 1:366-373,
1998).
Subsequent to glutamate addition, changes in O~I' are measured using the ET-
based
assay of 0'f. Mitochondria-defective cybrid cells that have a depressed O~I'
(Cassarino
et al., Biochem. Biophys. Res. Commun. 248:168-173, 1998) are also utilized in
addition
to primary neuronal cultures in order to provide a more extensive response to
agents
and/or conditions that are tested for their ability to dissipate or collapse
0'Y.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
118
Other examples of cells comprising one or more types of glutamate
receptors that are used in primary screens include cells that have been
genetically
engineered to express or overexpress one or more glutamate receptors. A number
of
mammalian cell lines have been manipulated to stably express glutamate
receptors in
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 NMDRAIA/2A and NMDAR1A/2B (Varney et al., J.
Pharmacol. Exp. Ther. 279:367-378, 1996); NMDAR2C, isoforms l, 2, 3 and 4
(Dagget et al., J. Neurochem. 71:193-1968, 1998); GluR3 (Varney et al., J.
Pharmacol. Exp. Ther. 285:358-370, 1998); and GluRlb and GluRSa (Lin et al.,
Neuropharmacology 36:917-931, 1997).
Secondary Screening Assa,
Compounds that prevent the prolonged collapse of 0'fm caused by high
[Ca'+;] in the primary assay are evaluated further in secondary assays,
including ROS
I 5 production, measurement of cytochrome c release and caspase-3 activation
as indicators
of apoptosis, and cell viability. In this way, "hits" identified in the FRET
O~fm assay
are further verified, and the mechanism by which the compound affects D~f",
can be
better defined. The rationale for these assays is based on evidence suggesting
that
compounds that can maintain mitochondria) integrity under conditions of
excitotoxicity
or oxidative stress may correspondingly decrease the release of apoptogens and
rescue
penumbra) neurons that are at risk of apoptotic death following transient
ischemia. The
following assays are described in more detail in copending U.S. patent
application
Serial No. 09/299,044, filed April 23, 1999.
Assay for Inhibition of Production of Reactive Oxy eg n Species Using
Dichlorofluorescin Diacetate: According to this assay, the ability of a
mitochondria
protecting agent of the invention to inhibit production of ROS intracellularly
may be
compared to its antioxidant activity in a cell-free environment. Production of
ROS may
be monitored using, for example by way of illustration and not limitation,
2',7'-
dichlorodihydroflurescein diacetate ("dichlorofluorescin diacetate" or DCFC),
a
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
119
sensitive indicator of the presence of oxidizing species. Non-fluorescent DCFC
is
converted upon oxidation to a fluorophore that can be quantified
fluorimetrically. Cell
membranes are also permeable to DCFC, but the charged acetate groups of DCFC
are
removed by intracellular esterase activity, rendering the indicator less able
to diffuse
back out of the cell.
In the cell-based aspect of the DCFC assay for inhibition of production
of ROS, cultured cells may be pre-loaded with a suitable amount of DCFC and
then
contacted with a mitochondria protecting agent. After an appropriate interval,
free
radical production in the cultured cells may be induced by contacting them
with iron
(III)/ ascorbate and the relative mean DCFC fluorescence can be monitored as a
function of time.
In the cell-free aspect of the DCFC assay for inhibition of production of
ROS, a mitochondria protecting agent may be tested for its ability to directly
inhibit
iron/ ascorbate induced oxidation of DCFC when the protecting agent, the
fluorescent
indicator and the free radical former are all present in solution in the
absence of cells.
Comparison of the properties of a mitochondria protecting agent in the
cell-based and the cell-free aspects of the DCFC assay may permit
determination of
whether inhibition of ROS production by a mitochondria protecting agent
proceeds
stoichiometrically or catalytically. Without wishing to be bound by theory,
mitochondria protecting agents that scavenge free radicals stoichiometrically
(e.g., on a
one-to-one molecular basis) may not represent preferred agents because high
intracellular concentrations of such agents might be required for them to be
effective in
vivo. On the other hand, mitochondria protecting agents that act catalytically
may
moderate production of oxygen radicals at their source, or may block ROS
production
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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
120
having such catalytic inhibitory characteristics may be a preferred agent for
use
according to the method of the invention
Mitochondria protecting agents that are useful according to the instant
invention may inhibit ROS production as quantified by this fluorescence assay
or by
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.
Assay for Mitochondria) Permeability Transition (MPT) Using 2.4-
Dimethylaminostyryl-N-Methylpyridinium (DASPMI): According to this assay, one
may determine the ability of a mitochondria protecting agent of the invention
to inhibit
the loss of mitochondria) membrane potential that accompanies mitochondria)
dysfunction. As noted above, maintenance of a mitochondria) membrane potential
may
be compromised as a consequence of mitochondria) dysfunction. This loss of
membrane potential or mitochondria) permeability transition (MPT) can be
quantitatively measured using the mitochondria-selective fluorescent probe 2-
,4-
dimethylaminostyryl-N-methylpyridinium (DASPMI).
Upon introduction into cell cultures, DASPMI accumulates in
mitochondria in a manner that is dependent on, and proportional to,
mitochondria)
membrane potential. If mitochondria) 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 mitochondria) 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.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
121
Assays of Apoptosis in Cells Treated with Mitochondria Protecting
A-gents: As noted above, mitochondria) 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. Mitochondria) dysfunction may be present in cells known or
suspected of
being derived from a subject with a mitochondria associated disease, or
mitochondria)
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.
Apoptosis and/or biochemical processes associated with apoptosis may
also be using one or more ''apoptogens," i.e., agents that induce apoptosis
and/or
associated processes when contacted with or withdrawn from cells or isolated
mitochondria. Such apoptogens include by way of illustration and not
limitation ( I )
apoptogens that are added to cells having specific receptors therefor, e.g.,
tumor
necrosis factor (TNF), Fast, glutamate and NMDA; (2) withdrawal of growth
factors
from cells having specific receptors for such factors, such factors including,
for
example, IL-3 or corticosterone; and apoptogens that may be added to cells but
which
do not require a specific receptor, including (3) Herbimycin A (Mancini et
al., J. Cell.
Biol. 138:449-469, 1997), (4) Paraquat (Costantini et al., Toxicology 99:1-2,
1995); (5)
ethylene glycols (http://www.ulaval.ca/vrr/rech/Proj/532866.html); (6) protein
kinase
inhibitors, such as, e.g.: Staurosporine, Calphostin C, d-erythro-sphingosine
derivatives,
Chelerythrine chloride, Genistein, I-(5-isoquinolinesulfonyl)-2-
methylpiperazine, KN-
93, Quercitin, N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide
and
caffeic acid phenethyl ester; (7) ionophores such as, e.g.: 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; (I1) 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,
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
122
Bleomycin sulfate, Hydroxyurea, Methotrexate, Mitomycin C, Camptothecin,
daunorubicin and intercalators such as, e.g., doxorubicin; (16) protein
synthesis
inhibitors such as, e.g., cyclohexamide, puromycin and rapamycin; (17) agents
that
affect microtubulin formation or stability such as, e.g., Vinblastine,
Vincristine,
colchicine, 4-hydroxyphenylretinamide and paclitaxel; ( 18) agents that raise
intracellular calcium levels by causing the release thereof from intracellular
stores, such
as, e.g., thapsigargin (Thastrup et al., Proc. Natl. Acad. Sci. U.S.A. 87:2466-
2470,
1990), thapsigargicin (Santarius et al., Toxicon 2:389-399, 1987) and
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. Natl. Acad. Sci. U.S.A. 9:4997-5002, 1998); and
(20)
calcium and inorganic phosphate (Kroemer et al., Ann. Rev. Physiol. 60:619-
642, 1998).
In one aspect of the apoptosis assays, cells that are suspected of
undergoing apoptosis may be examined for morphological, permeability or other
changes that are indicative of an apoptotic state. For example by way of
illustration and
not limitation, apoptosis in many cell types may cause altered morphological
appearance such as plasma membrane blebbing, cell shape change, loss of
substrate
adhesion properties or other morphological changes that can be readily
detected by
those skilled in the art using light microscopy. As another example, cells
undergoing
apoptosis may exhibit fragmentation and disintegration of chromosomes, which
may be
apparent by microscopy and/or through the use of DNA specific or chromatin
specific
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,
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
123
1999). These and other means for detecting apoptotic cells by morphologic,
permeability and related changes will be apparent to those familiar with the
art.
In another aspect of the apoptosis assays, translocation of cell membrane
phosphatidylserine (PS) from the inner to the outer leaflet of the plasma
membrane is
quantified by measuring outer leaflet binding by the PS-specific protein
annexin
(Martin et al, J. Exp. Med. 182:1545-1556, 1995; Fadok et al., J. Immunol.
1=18:2207-
2216, 1992.). In a preferred format, exteriorization of plasma membrane PS is
assessed
in 96-well plates using a labeled annexin derivative such as an annexin-
fluorescein
isothiocyanate conjugate (annexin-FITC, Oncogene Research Products, Cambridge,
MA).
In another aspect of the apoptosis assays, quantification of the
mitochondria) protein cytochrome c that has leaked out of mitochondria in
apoptotic
cells may provide an apoptosis indicator that can be readily determined (Liu
et al., Cell
86:147-157, 1996). Such quantification of cytochrome c may be performed
spectrophotometrically, immunochemically or by other well established methods
for
detecting the presence of a specific protein. Release of cytochrome c from
mitochondria in cells challenged with apoptotic stimuli (e.g., ionomycin, a
well known
calcium ionophore) can be followed by a variety of immunological methods.
Matrix-
assisted laser desorption ionization time of flight mass (MALDI-TOF)
spectrometry
coupled with affinity capture is particularly suitable for such analysis since
apo-
cytochrome c and holo cytochrome c can be distinguished on the basis of their
unique
molecular weights. For example, the SELDI system (Ciphergen, Palo Alto, USA)
may
be utilized to follow the inhibition by mitochondria protecting agents of
cytochrome c
release from mitochondria in ionomycin treated cells. In this approach, a
cytochrome c
specific antibody immobilized on a solid support is used to capture released
cytochrome
c present in a soluble cell extract. The captured protein is then encased in a
matrix of an
energy absorption molecule (EAM) and is desorbed from the solid support
surface using
pulsed laser excitation. The molecular weight of the protein is determined by
its time of
flight to the detector of the SELDI mass spectrometer.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
124
In another aspect of the apoptosis assays, induction of specific protease
activity in a family of apoptosis-activated proteases known as the caspases
(Thornberry
and Lazebnik, Science 281:1312-1316, 1998) is measured, for example by
determination of caspase-mediated cleavage of specifically recognized protein
substrates. These substrates may include, for example, poly-(ADP-ribose)
polymerase
(PARP) or other naturally occurring or synthetic peptides and proteins cleaved
by
caspases that are known in the art (see, e.g., Ellerby et al., J. Neurosci.
17:6165-6178,
1997). The labeled synthetic peptide Z-Tyr-Val-Ala-Asp-AFC, wherein "Z"
indicates a
benzoyl carbonyl moiety and AFC indicates 7-amino-4-trifluoromethylcoumarin
(Kluck
et al., 1997 Science 27:1132-1136, 1997; Nicholson et al., Nature 376:37-43,
1995), is
one such substrate. Another labeled synthetic peptide substrate for caspase-3
consists
of two fluorescent proteins linked to each other via a peptide linker
comprising the
recognition/cleavage site for the protease (Xu et al., Nucleic Acids Res.
26:2034-2035,
1998). Other substrates include nuclear proteins such as L11-70 kDa and DNA-
PKcs
(Rosen and Casciola-Rosen, J. Cell. Biochem. 64:50-454, 1997; Cohen, Biochem.
J.
326:1-16, 1997).
In another aspect of the apoptosis assays, the ratio of living to dead cells,
or the proportion of dead cells, in a population of cells exposed to an
apoptogen is
determined as a measure of the ultimate consequence of apoptosis. Living cells
can be
distinguished from dead cells using any of a number of techniques known to
those
skilled in the art. By way of non-limiting example, vital dyes such as
propidium iodide
or trypan blue may be used to determine the proportion of dead cells in a
population of
cells that have been treated with an apoptogen and a compound according to the
invention.
The person of ordinary skill in the art will readily appreciate that there
may be other suitable techniques for quantifying apoptosis, and such
techniques for
purposes of determining the effects of mitochondria protecting agents on the
induction
and kinetics of apoptosis are within the scope of the assays disclosed here.
Assay of Electron Transport Chain (ETCI Activity in Isolated
Mitochondria: As described above, mitochondria associated diseases may be
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
125
characterized by impaired mitochondria) 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 mitochondria) 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 -l=1:1090-1096, 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
mitochondria) function.
Furthermore, mitochondria) function may be monitored by measuring the
oxidation state of mitochondria) cytochrome c at 540 nm. As described above,
oxidative damage that may arise in mitochondria associated diseases may
include
damage to mitochondria) components such that cytochrome c oxidation state, by
itself
or in concert with other parameters of mitochondria) function including but
not limited
to mitochondria) oxygen consumption, may be an indicator of reactive free
radical
damage to mitochondria) components. Accordingly, 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.
For example by way of illustration and not limitation, 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
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
126
through a sealed rubber stopper. The reaction may be initiated by addition of
a cell
homogenate or, preferably a preparation of isolated mitochondria, via
injection through
the rubber stopper. This assay, or others based on similar principles, may
permit
correlation of mitochondria) respiratory activity with structural features of
one or more
mitochondria) components. In the assay described here, for example, a defect
in
complex IV activity may be correlated with an enzyme recognition site.
Tertiary Screening Assays
Compounds that possess the desired activity profile in secondary in vitro
assays are tested for in vivo efficacy in the rodent middle cerebral artery
occlusion
(MCAO) model of transient focal ischemia that is reported to produce ischemia
analogous to MCAO branch occlusion in humans (Longa et al., Stroke 1:84-91,
1989).
Initially, test compounds are administered by a continuous intravenous
infusion before
and during the ischemia/reperfusion period to ensure the greatest chance for
experimental success. Once efficacy is established, experiments are conducted
in which
1 S efficacy is assessed as a post-treatment using single and multiple drug
administration
regimens. The efficacy of the test compounds is directly assessed by measuring
the
reduction of neuronal loss in the infarcted brain region using techniques such
as
magnetic resonance imaging. Other additional endpoints are then measured,
including
reduction of brain lactate production as a consequence of the switch from
aerobic to
anaerobic metabolism after oxygen deprivation, reduction in DNA, protein and
lipid
oxidation products.
EXAMPLE 10
ET-BASED ASSAYS FOR MONITORING FUSION OF SUBCELLULAR COMPARTMENTS
Assays utilizing energy transfer can be used to monitor the fusion of
subcellular compartments such as, e.g., organelles. For example, mitochondria
undergo
changes, including fission and fusion, and the latter process involves
apparently
coordinated rearrangements of internal elements (i.e., the inner membrane,
cristae, etc.)
(for a review, see Bereiter-Hahn and Voth, Microscopy Research and Technique
27:198-219, 1994). Such changes are believed to be important for various
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
127
developmental processes. In a variety of organisms including yeast such as C.
cerevisiae, insects such as D. melanogaster, invertebrates such as C. elegans,
and
mammals such as H Sapiens, fusion of mitochondria is mediated by GTPase
proteins
generally known as "mitofusins" (see Hales et al., Cell 90:121-129, 1997;
Hermann et
al., J. Cell. Biol. 1=13:359-373, 1998; and published PCT patent application
WO
98/55618). Mutations in the fuzzy onions (fzo) gene, which encodes a mitofusin
in D.
melanogaster, impair spermatogenesis and renders male insects sterile (Hales
et al., Cell
90:121-129, 1997).
Accordingly, in certain embodiments the present invention provides a
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 mitochondria) 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 first and second samples with one another in
the
absence and presence of a candidate agent under conditions and for a time
sufficient to
permit mitochondria) 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.
In those certain preferred embodiments wherein the invention is directed
to a method for identifying an agent that alters mitochondria) fusion, neither
the ET
donor molecule nor the ET acceptor molecule is endogenous to mitochondria, and
the
ET donor and the ET acceptor each localize independently of one another to the
same
submitochondrial site or to acceptably adjacent submitochondrial sites as
provided
herein. Typically, based upon the teachings provided herein, a person having
ordinary
skill in the art can readily determine when a candidate agent alters
mitochondria) 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.
As noted above, conditions permissive for mitochondria) fusion events are
known in the
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
128
art, such that those having ordinary skill in the art can readily determine
what are
suitable conditions for conducting the instant assay method without undue
experimentation. By way of illustration and not limitation, such conditions
may include
those that permit fusion of isolated mitochondria, which refers to
mitochondria that
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 mitochondria)
to
undergo fusion within cells.
It is desirable to develop novel antibiotics or pesticides that function by
selectively inhibiting mitofusin activity in undesirable insects or eukaryotic
parasites
but have minimal or no effect on the mitofusin of desirable insects or plants
or on
mammalian hosts including humans. It is also desirable to identify and
characterize
agents that stimulate or inhibit intracellular mitochondria) fusion events for
the
treatment of human diseases. The present invention can be used to achieve
these goals
in the following manner.
In general, a first group of mitochondria is preincubated with a donor
compound, and a second group of mitochondria is incubated with an appropriate
acceptor compound. Coincubation of the first and second group of mitochondria
will
result in fusion of individual mitochondria from each set, in which case the
donor and
acceptor compounds will achieve proximity to each other. Thus, mitochondria)
fusion
will lead to energy transfer that can be measured according to the present
disclosure. If
an agent that stimulates or inhibits mitochondria) 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.
EXAMPLE 11
ET-BASED ASSAYS FOR MONITORING LOCALIZATION OF AGENTS
TO SPECIFIC SUBCELLULAR SITES
Assays utilizing energy transfer can be used to monitor the influx or
efflux of agents into a specific subcellular compartment within isolated
organelles or
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
129
intact cells; in the latter case, such assays can be used to estimate
pharmacokinetic
properties of candidate therapeutic agents. For example, agents comprising
tertramethylrhodamine (TMR) or related moieties have been described. For
example,
oligonucleotides that are 5'-end labeled with TMR are available from Genomyx
Corp.
(Foster City, CA), and dideoxynucleotides conjugated to rhodamine or
dichlororhodamine moieties are available from the Perkin-Elmer Corp. (Norwalk,
CT).
General methods for preparing conjugates comprising NAO- or JC-1-based
moieties are
described in published PCT patent application WO 98/17826. Mitochondria)
uptake of
such agents can be evaluated using the present invention as follows.
The uptake of agents comprising tertramethylrhodamine (TMR) or
related moieties into mitochondria can be monitored by preincubating
mitochondria or
cells containing mitochondria with a donor compound such as NAO, MitoTracker~
Green FM or MitoFluorTM Green for a period of time, after which the TMR-
conjugated
agent of interest is added. If the agent is taken up by mitochondria, the TMR
or TMR-
like portion thereof will act as an acceptor for energy emitted from the donor
compound. Uptake of the agent can thus be followed as a function of either
decreasing
emission from the donor or increasing emission from the TMR or TMR-like
moiety.
Similarly, the uptake of agents comprising NAO or NAO-like moieties
into mitochondria can be monitored by preincubating mitochondria or cells
containing
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.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
130
EXAMPLE 12
FRET-BASED ASSAY OF Di4! IN PERMEABILIZED CELLS
In the following experiments, FRET-based assays of Dye 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.
Control
experiments were performed to demonstrate that the same response to different
concentrations of calcium is seen in permeabilized cells in the presence or
absence of
ionomycin, which confirms that calcium ions freely enter the permeabilized
cells to at
least the same degree as ionomycin-treated cells.
Another noteworthy difference between the protocols of the assays
described in this example and those described above is that cells not
contacted with both
the ET donor and ET acceptor compounds at the same time, as in Examples 5-7.
Instead, cells were initially contacted with NAO only (0.04 uM; 5 minutes),
rinsed 3
times at 37°C in HBSS buffer (although many other buffers are suitable
for these
rinses), and then transferred to a plate reader, i.e., an instrument capable
of reading the
signal produced due to energy transfer in the assay, preferably an automated
or semi-
automated instrument such as a FLIPRTM instrument, which is described above.
Once in the plate reader, the signal due to NAO was monitored for about
1 minute before addition of TMR as the second member of the ET pair. As
described
above and according to non-limiting theory, TMR accumulates in mitochondria as
a
function of Dy and quenches, via FRET, the fluorescent signal from NAO. As Dyr
changes in response to various compounds or conditions, the concentration of
TMR in
mitochondria changes in a corresponding manner, as reflected by changes in the
signal
corresponding to NAO quenching.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
131
Typically, the average of several readings during this interval was taken
for analysis and is labeled "Q" in Figure 16. The quenching of NAO by TMR was
monitored in real time to ensure that equilibrium was reached before the
addition of test
compounds and/or other agents. This steady state is labeled "R" in Figure 16.
Cells
were washed and all reagents prepared in Hanks Balanced Salt Solution (HBSS)
containing 20 mM HEPES buffer. Test compounds were then added and allowed to
equilibrate for at least 5 minutes; the average of several readings that
correspond to the
latter interval is labeled as "S" in Figure 16. In the final phase of the
assay (labeled "T"
in Figure 16), Dy was collapsed by the direct addition of Caz+ to
permeabilized cells.
This step differs from the protocols of the preceding examples, wherein Ca2+-
mediated
changes in Dyr were induced in intact (nonpermabilized) cells by an ionophore
(e.g.,
ionomycin) that promoted entry of high concentrations of CaZ+ into the cytosol
from the
extracellular environment.
In the protocol used in this example, 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.
Another modification of the assay in this Example involved data
handling. The data were analyzed by dividing the signal recovered after Ca2+
addition
(the difference of T-S) by the signal that was initially quenched by TMR (the
difference
of Q-R). These mathematical manipulations yielded a ratio different from those
presented in Example 5 (i.e., "Dyr-Dissipating Value" and "Efficacy Index").
Data such
as those presented in Figures 16 and 17 can be used to derive a "Ratio of
Recovery of
Initial Quenching" (hereafter, "RRIQ") according to the following formula:
RRIQ-LT_S]/[Q-R]
Agents that inhibit or block Ayr collapse have an RRIQ that is less than
that of control samples exposed to Ca2+ but not treated with such agents.
Conversely,
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
132
compounds that induce or enhance Dyr collapse have an RRIQ that is greater
than that
of untreated controls.
It is also possible to evaluate the direct effect of compounds on Dyr by
examination of the values corresponding to the difference between R and S (i.
e., R-S).
Hyperpolarizing agents such as oligomycin increase TMR uptake, and hence
increase
NAO quenching, which results in a positive value for R-S. Conversely,
compounds
that directly dissipate Dyr yield a negative value for R-S.
Validation studies indicated that Ca2+ showed a dose-dependent
response, where high Ca2+ levels caused Dyr to collapse to the initial levels
with only
minimal recovery of Dy. However, intermediate Ca2+ levels permitted some
recovery
of Dyr, which is often lost as mitochondria undergo secondary permeability
transition.
That this is indeed secondary transition was supported by the observation that
such
transition was inhibited by cyclosporin A and bongkrekic acid. In many cases,
data
from such situations were best analyzed by summing the area under the curve
(AUC)
for a specific duration after Ca'+ addition, rather than by using an average
response for a
specified interval.
In addition to the ability to monitor secondary OW collapse due to
permeability transition, the protocol used in the instant Example also permits
identification of agents that influence mitochondrial Ca2+ uptake, such as RU-
360. In
this case, the initial depolarization upon Ca2+ addition was diminished
compared to
untreated controls. Analysis was accomplished by comparing the maximum peak
height immediately after Ca2+ addition.
As shown in Figure 17, permeabilized cells treated with calcium alone
(i.e., with no ionophore present) underwent a concentration-dependent response
to
calcium ions, leading to a collapse of Dy at 100 ~M Ca'+ that was roughly
equivalent,
in terms of both the extent of response and time course, to that seen in cells
treated with
the Dy-collapsing agent CCCP. The data shown in Figures 16 and 17 were
generated
using SH-SYSY cells permeabilized by digitonin (0.008% v/v) obtained from
Sigma
(St. Louis, MO). The cells were grown in media comprising 125 mM KCI, 2mM
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
133
K~HP04, 5 mM HEPES, 4 mM MgClz, 1 mM malate, 1 mM succinate, 1 mM
glutamate, 1 uM EGTA, pH 7Ø
In Figure 18, the RRIQs derived from the data shown in Figures 16 and
17 are plotted as a function of the concentration (log M) of calcium ions
(Caz+). This
Concentration Response Curve (hereafter, "CRC") yields an ECso of 56.5 ~M for
Ca'+
in permeabilized cells.
Data presented in Example 3 demonstrated that, in intact (i.e.,
nonpermeabilized) cells, ruthenium red (an inhibitor of the mitochondria)
calcium
uniporter) modulated [ionomycin + Ca'+]-induced collapse of Dy (e.g., Figure
8). In
these experiments (the instant Example 12), by way of contrast, an ionophore
(ionomycin) was used to facilitate the entry of Caz+ into the cytosol of
cells. Figure 19
shows a CRC of RU-360 (concentrations from 0 to 25 nM), an inhibitor of the
calcium
uniporter that is more specific than ruthenium red, in digitonin-permeabilized
cells
treated with Ca'-~. The results yielded an ECSO of 11 nM for RU-360 in
permeabilized
1 S and Ca2+ treated cells. TJnlike the results presented in Figures 18 and
20, wherein
RRIQs are plotted as a function of the concentration of a candidate or control
agent, the
responses in Figure 19 were summarized by calculating the area under the
respective
curves (see Example 4 and Table 6) for each concentration of RU-360, rather
than
RRIQ values.
Data also presented in Example 3 show that in intact cells, cyclosporin A
(hereafter, "CsA") modulated [ionomycin + Ca2+]-induced collapse of Dyr (e.g.,
Figure
9) at a concentration of 10 ~.M. In digitonin-permeabilized cells treated with
CsA, CsA
moderated the Ca2+-induced Dye collapse with an ECSO of 0.31 pM (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.
The results presented in this Example demonstrate various useful
embodiments of the assays of the invention that utilize permeabilized cells.
According
to one such embodiment, a range of distinct permeabilization conditions is
employed to
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
134
determine those wherein the plasma membrane selective permeability of a cell
is
compromised while organellar membranes (e.g., one or both of the membranes
surrounding mitochondria and/or chloroplasts) retain their selective
permeability.
Permeabilization conditions such as those described in the preceding
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. In the case of objective (3), the goal of the assay may be to select
or identify,
from a group of candidate agents, active compounds that are antagonists or
agonists of
agents that influence the activity of certain organelles and organellar
components. In
the latter situation, permeabilization of cells allows one to contact
organelles with (i)
agents that influence the activity of certain organellar components and (ii)
one or more
candidate agents, with the desirable features of contacting organelles with
both (i) and
(ii) at the same time and with a minimum of manipulation of the cells used in
the assay;
such features are particularly useful in high throughput (HTS) assays. As an
example of
the desirable aspect of achieving objective (3) in such assays, the results
presented
herein demonstrate the concomitant contacting of mitochondria in permeabilized
cells
with Ca2+, 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. Within certain ranges of
permeabilization
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
135
conditions, objectives (1), (2) and (3) can all be realized through one set of
permeabilization conditions; such conditions are particularly useful in high
throughput
(HTS) assays wherein it is desired to investigate the effects of various
combinations of
two or more molecules known or suspected to influence the activity of certain
organelles and organellar components, optionally in further combination with
one or
more compounds known or suspected to influence (e.g.,. enhance or decrease or
otherwise regulate the activity of) such molecules.
The results presented above derived from cells exposed to calcium ions
(Ca'+) and RU-360, an inhibitor of the mitochondrial calcium uniporter (Figure
19)
illustrate an embodiment of the assays of the invention in which the
permeabilization
treatment is such that the plasma membrane has lost its capacity to maintain
selective
permeability, but intracellular (e.g., organellar) membranes remained
selectively
permeable. That is, under the appropriate permeabilization conditions, Ca'~
diffused
across a permeabilized plasma membrane into the cytosol, but Ca'+ uptake into
mitochondria was inhibited by RU-360 in a concentration-dependent manner. Thus
mitochondrial membranes were not permissive for CA'y diffusion and apparently
through the activity of the calcium uniporter.
EXAMPLE 13
FRET-BASED ASSAY OF Dill IN PERMEABILIZED CELLS
2O USING REDUCED ET MOLECULE CONCENTRATIONS
In this example, FRET-based assays of Dy 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. According to non-limiting theory, the use of lower
concentrations of the
ET donor and acceptor molecules NAO and TMR avoids potential self quenching by
the potentiometric dye, and also avoids undesirable dissipation of Dyr as the
cationic
dye enters the mitochondrial matrix.
FRET methods using digitonin-permeabilized SH-SYSY cells were
essentially as described above in Example 12 and Figure 16, with exceptions as
noted
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
136
herein. All reagents were from Sigma (St. Louis, MO) unless otherwise stated.
Briefly,
96-well assay plates containing SYSY 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 Ca2+ (35 ~M or 50 ~M) was
added to collapse mitochondria) membrane potential and readings were taken for
an
additional 5-10 minutes.
Figure 21 shows the superimposed FRET RFU time course plot obtained
when various concentrations of oligomycin, a specific inhibitor of ATP
synthase, was
the added mitochondrially active compound. By way of non-limiting theory,
mitochondria) inner membrane hyperpolarization that was observed following
exposure
of permeabilized cells to oligomycin (Fig. 21 A) resulted from inhibition of
ATP
synthase and the consequent inhibition of normal Dyr dissipation via ADP
phosphorylation. Increased quenching of NAO by TMR was thus observed in the
presence of oligomycin in a dose-dependent manner between 5 and 12 minutes
(EC50 =
0.25 fig/ ml). Fig. 21B shows a CRC generated by calculating R-S, as described
above
in Example 12, for each oligomycin concentration (data points are shown + SEM,
RZ =
0.4123).
According to similar reasoning, exposure of permeabilized cells in the
FRET assay to an excess of ADP should result in a transient loss of Dyr as
mitochondria) membrane potential is dissipated by ATP synthase-mediated
phosphorylation of ADP. Such a transient loss of Ayr was observed when the
added
mitochondrially active compound was 6.25- 50 ~M ADP (Fig. 22A, EC50 = 12.1
~M).
Below 6.25 ~.M, the change in Dyr was indistinguishable from control groups
that were
exposed to buffer alone (ANOVA F = 36.4). The CRC plot as a function of ADP
concentration is shown in Fig. 22B.
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
137
Figure 23 shows the superimposed FRET RFU time course plot obtained
when various concentrations of bongkrekic acid (BKA), a specific inhibitor of
the
mitochondria) adenine nucleotide translocase, was the added mitochondrially
active
compound. By way of non-limiting theory, mitochondria) inner membrane
hyperpolarization that was observed following exposure of permeabilized cells
to BKA
(Fig. 23A) resulted from inhibition of ADP entry into the mitochondria)
matrix, and the
consequent inhibition of normal Dye dissipation via ADP phosphorylation. BKA
is also
believed to forestall mitochondria) permeability transition resulting from
Caz+ load.
Increased quenching of NAO by TMR was thus observed in the presence of BKA in
a
dose-dependent manner between 5 and 11 minutes (EC50 =- 0.09 ~tM). Following
addition of Ca'l to the permeabilized cells, BKA potentiated the extent of Dyr
recovery
and also moderated the secondary loss of Dyr (Fig. 23A), due probably to
permeability
transition. Fig. 23B shows a CRC generated by calculating R-S, as described
above in
Example 12, for each BKA concentration (data points are shown + SEM, R' =
0.5822).
1 S 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
mitochondria) membrane that derives from a pH gradient, was the added
mitochondrially active compound. By way of non-limiting theory, mitochondria)
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
mitochondria) membrane proton-motive force (e.g., electron transport).
Increased
quenching of NAO by TMR was thus observed in the presence of 0.09-0.75 ~M
nigericin at timepoints between 6 and 12 minutes. At higher nigericin
concentrations
(e.g., > 3 ~M), there was a steep loss of NAO quenching (Fig. 24A), presumably
(and
according to non-binding theory) due to inability of electron transport to
compensate for
OpH dissipation. Fig. 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 O~r collapsed (> 1.5~M) and at which mitochondria)
inner
CA 02375542 2001-12-13
WO 00/79274 PCT/US00/17380
138
membrane hyperpolarization was detectable (<0.75 pM) using the FRET assay
conditions described herein (data points are shown + SE, ANOVA P <0.0005).
From the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration,
various modifications may be made without deviating from the spirit and scope
of the
invention. Accordingly, the invention is not limited except as by the appended
claims.
