JP2003501024A - Assay for caspase activity using green fluorescent protein - Google Patents

Abstract

(57) SUMMARY Substrates and assays for the identification of activators and inhibitors of caspases are provided. The substrate is a fusion protein containing two green fluorescent proteins (GFP) with a linker peptide containing at least one caspase cleavage site. The complete fusion protein shows fluorescence resonance energy transfer (FRET) between GFP. When the linker peptide is degraded by caspase, two GFPs are separated, and FRET is attenuated.

Description

Detailed Description of the Invention

[0001]     (Cross-reference of related applications)   Not applicable.

[0002]     (Mention on federally sponsored R & D)   Not applicable.

[0003]     (See microfish appendix)   Not applicable.

TECHNICAL FIELD The present invention relates to methods for determining the activity of caspases. The method involves fluorescence resonance energy transfer between two green fluorescent proteins linked as a fusion protein having a caspase cleavage site between them.
gy transfer) is used. The method can be used to identify substances that are activators or inhibitors of caspase activity, either in viable cells or in vitro.

BACKGROUND OF THE INVENTION Apoptosis is characterized by a series of well-defined morphological and biochemical changes that inevitably lead to cell death (Kerr et al., 1972, Br. J. Canc.
er 26: 239-257: Vaux et al., 1994, Cell 76: 777.
-779; Steller, 1995, Science 267: 1445-1.
449). Apoptosis occurs in developing morphogenesis, in the removal of exhausted, unwanted, or irreparable injured cells and in response to pathogenic infection (Kerr et al., 1972, Br. J. et al. Cancer 26: 239-2
57: McConkey et al., 1996, Mol. Aspects Med. 17
: 1-110; Steller, 1995, Science 267: 1445.
-1449; Thompson, 1995, Science 267: 1456.
-1462; Uren & Vaux, 1996, Pharmacol. Ther.
72: 37-50). Apoptosis proceeds through a series of highly regulated biochemical events and many components of the cell death pathway have been identified. Among the key biochemical mediators of apoptosis are the proteolytic enzymes known as caspases, which target specific target proteins with either structural, regulatory, or housekeeping functions. By degrading it acts to disrupt normal cell homeostasis (Thornberry & Lazebnik,
1998, Science 281: 1312-1316; Thornberr.
y, 1997, Br. Med. Bull. 53: 478-490; Nichol.
Son & Thornberry, 1997, Trends Biochem. S
ci. 22: 299-306). This results in arrest of normal cell function, disruption of genomic and cellular structural elements, and packaging of cellular components into apoptotic debris for uptake by other cells (Nicholson & Thorn.
Berry, 1997, Trends Biochem. Sci. 22: 219
9-306; Rosen & Casciola-Rosen, 1997, J. Am. Ce
ll. Biochem. 64: 50-54).

Caspases constitute a large family of proteins that can be distinguished according to their substrate specificity (Thornberry, 1997, J Biol Chem).
272: 17907-17911). So far 12 caspases have been identified in human cells. All caspases are synthesized as inactive proenzymes and can be rapidly activated through proteolytic maturation (Thornbe).
rry & Lazebnik, 1998, Science 281: 1312-1.
316; Thornberry, 1997, Br. Med. Bull. 53: 4
78-490). Members of the caspase family can be grouped into three functional subgroups based on substrate specificity, defined by the positional scanning combinatorial substrate method (Rano et al., 1997, Chem. Biol. 4).
149-155; Thornberry et al., 1997, J. Am. Biol. Che
m. 272: 17907-17911). Major apoptotic effectors (
II, including Caspase-2, -3, and -7, and C. elegans CED-3
Group caspases) are motifs that are degraded during apoptosis found in cleavage site known most _proteins, having specificity for _{[P 4] DExD [P 1} ] (Nicholson & Thornberry, 1997, Trend
s Biochem. Sci. 22: 299-306). On the other hand, the specificity of group III caspases (caspase-6, -8, -9, and -10, and CTL-derived granzyme B) is comparable to that of other caspase proenzymes including group II (effector) family members. It is [P ₄ ] (I, V, L) ExD [P ₁ ] corresponding to the activation site at the junction between the large subunit and the small subunit. This and other evidence indicate that group III caspases function as upstream activators of group II caspases in proteolytic cascades that amplify death signals.

Since caspases play a central role as death effector molecules, they have recently received great attention as therapeutic targets for various disease processes (eg, acute neurodegeneration, cardiac ischemia, liver failure). Nicholson, 1996, Nat. Bi
otechnol. 14: 297-301). Unfortunately, methods for monitoring caspase activity, at least with respect to cell-based systems, are often several steps far from proteolysis caused by active caspases, downstream biochemical events (eg, , DNA fragmentation, membrane remodeling)
Mainly limited to the analysis of (Darzynkiewicz & Traganos
, 1998, Adv. Biochem. Eng. Biotechnol. 62:
33-73). Furthermore, sensitive substrates that are cell-permeable and specific for different caspase subgroups have not yet been developed. Therefore, there is a need for substances that are sensitive, specific to a particular caspase, provide a real-time readout, and can act intracellularly.

GFP-based reporter strategies have been used in numerous cellular systems to monitor transcriptional activation, subcellular localization, and protein trafficking (Mis).
teli et al., 1997, Nat. Biotechnol. 15: 961-964
Tsien, 1998, Annu. Rev. Biochem. 67: 509-
544). Applicability as an in vitro substrate for protease assays has also been demonstrated (Pollock & Heim, 1999, Trends Ce.
ll Biol. 9: 57-60; Mitra et al., 1996, Gene 173.
: 13-17; Heim et al., 1996, Curr. Biol. 6: 178-18
2). Xu et al., 1998, Nucl. Acids Res. 26: 2034-2
035 (Xu) disclosed the use of a fusion protein comprising a blue fluorescent protein and a green fluorescent protein linked by an 18 amino acid peptide containing a caspase-3 cleavage site. This fusion protein is used for fluorescence activated cell sorting (FACS).
Was used to monitor activation of caspase activity by the use of H. and the detection of fragments of the fusion protein by Western blot. Xu did not disclose a method of high throughput screening to identify inhibitors of caspases.

Heim & Tsien, 1996, Current Biology 6: 1.
78-182 disclosed the use of a fusion protein containing two GFPs separated by a 25 amino acid linker which can be degraded by trypsin or enterokinase. International Patent Publication WO 97/28261 disclosed the use of a fusion protein containing two GFPs separated by a linker that can be degraded by a protease. Mitra et al., 1996, Gene 173: 13-17 disclosed the use of a fusion protein containing two GFPs separated by a 20 amino acid linker that can be cleaved by Factor Xa.

DISCLOSURE OF THE INVENTION The present invention provides a method of monitoring caspase activity by utilizing fluorescence resonance energy transfer between two green fluorescent proteins linked as a fusion protein having a caspase cleavage site between them. Regarding The method is rapid, sensitive, and generally operates at the level of caspase activity, ie, proteolysis of caspase substrates, rather than at the level of downstream events. For example, the method can be performed in vitro, using cell-free extracts or purified components, or can be performed in vivo, ie in living cells. The method is adapted to provide an assay suitable for identifying activators or inhibitors of caspase activity.

(Brief Description of Drawings) FIGS. 1A and 1B are diagrams showing fluorescence resonance energy transfer between mutant GFP variants in a recombinant group II caspase substrate. FIG. 1A: Donor GFP (W at 434 nm
When 1B) is excited, its released energy (476 nm) is transferred to the acceptor GFP (TOPAZ) and re-emitted at 527 nm. This energy transfer depends on the physical proximity of the two GFP proteins. Caspase-mediated degradation of the linker between GFPs eliminates energy transfer through separation of GFP molecules. FIG. 1B: Below the schematic of the tandem GFP protein, the primary amino acid sequence of the linker for each of the constructed fusion proteins (WT, WT-1x, WT-2x, and WT-2xL) is abbreviated. Note that the WT fusion contains a short 6 amino acid linker that does not contain the caspase tetrapeptide degradation motif. The WT-1x and WT-2x proteins contain single and two tandem group II caspase degradation motifs (DEVD (
It contains SEQ ID NO: 9)). WT-2xL contains a two tandem DEVD (SEQ ID NO: 9) degradation motif flanked by 6 amino acid SG repeats designed to confer flexibility and to facilitate access to the linker. The amino acid sequence shown for the WT linker is SEQ ID NO: 1, the amino acid sequence shown for the WT-1x linker is SEQ ID NO: 2, the amino acid sequence shown for the WT-2x linker is SEQ ID NO: 3, the WT-2xL linker is shown. The amino acid sequence is SEQ ID NO: 4.
Is.

2A-B show that FRET efficiency is a function of linker length.
Lysates of Hela cells transiently transfected with constructs encoding WT, WT-1x, WT-2x, or WT-2xL fusion proteins were assayed for fluorescence. FIG. 2A shows a lysate emission scan (450-600 nm) following excitation at 434 nm. FIG. 2B shows the measurement of FRET by ratiometric analysis (fluorescence emitted by acceptor (TOPAZ
, 527 nm) divided by the fluorescence emitted by the donor (W1B, 476 nm) (n = 2)).

FIG. 3 shows that a tandem GFP substrate containing multiple DEVD (SEQ ID NO: 9) motifs is degraded by caspase-3 more efficiently than a substrate containing a single DEVD (SEQ ID NO: 9) motif. It is a figure which shows that. The WT-1x protein and WT-2x protein labeled with translated ^[3 5 S] methionine in vitro, 1 hour at 37 ° C. with purified recombinant caspase-3 for various concentrations were incubated. Degraded proteins were separated by SDS polyacrylamide gel electrophoresis and quantified by gel autoradiography and scanning densitometry. The reaction was carried out using a substrate concentration well below Km, where the appearance of the product was the first-order process. S _t / S ₀ = e ^{−kobs * t} (where S _t is the concentration of the substrate remaining at time t, S ₀ is the initial substrate concentration, and k _obs =
from k _cat ^* [enzyme] a / _{K m)} the relationship, values were calculated kcal / Km.

FIG. 4A-B shows that apoptosis is tandem GFP containing DEVD (SEQ ID NO: 9).
It is a figure which shows that it brings about decomposition | disassembly of a board | substrate. Figure 4A shows the WT construct, WT-1x.
3 shows Western blot analysis of tandem GFP substrate in lysates prepared from Hela cells transfected with the construct, or with the WT-2x construct.
Apoptosis was induced by treating cells with 1 μM staurosporine for 4 hours. FIG. 4B shows FRET from cells treated with staurosporine in the presence (+) or absence (−) of a non-specific caspase inhibitor (zVAD-fmk).
The measurement is shown. Note that FRET is attenuated only in apoptotic extracts containing the caspase-3-sensitive tandem GFP substrate.

FIG. 5 is a diagram showing direct fluorescence quantification of caspase inhibition in whole cells. W
Hela cells overexpressing T-1x tandem GFP substrate (clone HETON
-WT-1x-57) was simultaneously treated with staurosporine to induce apoptosis and various concentrations of zVAD-fmk for 4 hours. After replacing the medium with PBS, cells were assayed for FRET (530em / 490em). 0
． The titration points of 01 μM and 10,000 μM were obtained respectively for the maximum (DMS
The ORET medium alone) and the minimum (staurosporine alone) FRET ratio values are shown.

6A-B show high throughput of caspase inhibitors in multi-well microtiter plates containing either a single compound (FIG. 6A) or drug mixture (10 compounds per well) (FIG. 6B). It is a figure which shows screening. Duplicate 96-well plates were co-treated with staurosporine and drug (s) for 4 hours. ZVAD-fmk at three concentrations (2.5 μM
, 25 μM, and 250 μM) were randomly added to 2 wells of each plate.
The experiment was performed blindly so that the zVAD-fmk well location was not revealed until the data was processed. Black and gray bars are 250 μM and 25 μM, respectively
The location of wells containing M zVAD-fmk is shown. False positive hits are indicated by the shaded bars. Column 1 (rows AH) represents uninduced non-apoptotic control wells.

7A-B shows the nucleotide sequence (FIG. 7A, SEQ ID NO: 5) and amino acid sequence (FIG. 7B, SEQ ID NO: 6) of wild-type green fluorescent protein.

[0018]     (Detailed Description of the Invention)   The following is for purposes of the present invention.

“Green fluorescent protein (GFP)” means any 150 consecutive amino acids
At least 8 with a continuous stretch of the amino acid sequence of wild-type GFP (SEQ ID NO: 6)
It is a fluorescent protein with 5% amino acid sequence identity.

“Ultra-bright green fluorescent protein (GFP)” was engineered to contain amino acid changes from wild-type GFP that result in enhanced release characteristics compared to wild-type GFP. It is GFP. Super bright GFP is Y66
Has no H change. Super bright GFP has a fluorescence quantum yield of at least 0.39. Examples of super bright GFP are W1B; TOPAZ; 10C; 10C Q69K
W7; W1C; wild type GFP with S65G change, S72A change, and T203F change (SEQ ID NO: 6); and S65G change, S72A change, and T203
Wild type GFP with H change (SEQ ID NO: 6).

A “high-throughput screen” refers to a large number of substances that are suspected of having the desired properties, eg, activators or inhibitors of caspases, and such substances actually exhibit such properties. A method of identifying an activator or inhibitor of a biological activity, eg, an activity of caspase, which is tested for its presence or absence. High-throughput screening is performed to determine if any of the substances have the desired properties, in order to determine if at least 5,000, preferably at least 10, per 24 hours.
It comprises testing 000, more preferably at least 100,000 substances. The method of high-throughput screening is less than 250 μl, preferably less than 25 μl, more preferably less than 10 μl in a microtiter-like plate having at least 96, preferably at least 384, more preferably 1,536 wells per plate. Performed in a volume of.

“Ratiometric analysis” means the fluorescence emitted by an acceptor green fluorescent protein (eg TOPAZ, 52) after excitation of the donor green fluorescent protein.
7 nm) the fluorescence emitted by the donor green fluorescent protein (eg W1B,
Fluorescence resonance energy transfer (FR) by determining the ratio divided by 476 nm).
ET).

“Fusion protein” refers to a polypeptide comprising an amino acid sequence in which two green fluorescent proteins (GFP) are joined by a linker consisting of a short amino acid stretch containing at least one caspase cleavage site. One GFP is the donor GFP and the other GFP is the acceptor GFP. Fluorescence resonance energy transfer from donor GFP to acceptor GFP when the linker is complete (
FRET) can occur, but the two GFPs are different GFPs so that FRET disappears or is greatly diminished when the linker is degraded.

“Caspase inhibitor” refers to a substance that reduces the proteolytic activity of a caspase protein. The caspase inhibitor need not have an effect on the amount of caspase protein produced by the cell. In addition, the caspase activator is
It does not necessarily act at the level of proteolysis by caspases. The caspase activator may be one that reduces the proteolytic activity of the caspase protein by acting at the level of upstream events that result in activation of the caspase. For example, the caspase inhibitor may suppress the release of cytochrome c from mitochondria, thereby suppressing the activation of caspase.
The method of the invention provides a means for identifying caspase inhibitors. After identification
Such inhibitors can be further characterized in terms of the level at which they act. One possibility would be to test the identified inhibitors in vitro, ie in a cell-free system containing purified caspase and GFP fusion protein substrate. If the inhibitor can suppress the loss of FRET from the fusion protein under such conditions, the inhibitor acts at the level of proteolysis by caspases. Other such secondary tests will be apparent to those of skill in the art.

“Caspase activator” refers to a substance that increases the proteolytic activity of a caspase protein. The caspase activator need not have an effect on the amount of caspase protein produced by the cell. Also, caspase activators do not necessarily act at the level of proteolysis by caspases. Caspase activators can increase the proteolytic activity of caspase proteins by acting at the level of upstream events that lead to activation of caspases. As with the caspase inhibitors, the level at which the caspase activator acts can be determined by a suitable in vitro assay.

The present invention uses fluorescence resonance energy transfer (FRET). What is FRET
This is a process of nonradiative transfer of energy from an excited donor fluorescent reagent to an acceptor fluorescent reagent due to long-range dipole-dipole coupling between molecules. F
RET typically occurs at a distance of about 10 to 100Å, and the emission spectrum of the donor reagent and the absorption spectrum of the acceptor reagent are appropriately overlapped, and the quantum yield of the donor and the absorption coefficient of the acceptor are Needs high enough. Furthermore, the transition dipoles of the donor and acceptor fluorescent reagents must be in the proper orientation with respect to each other. For a review of FRET and its application to biological systems, see, eg, Cleggg, 1995, Current Opini.
ons in Biotechnology 6: 103-110; Wu & Br.
and 1994, Anal. Biochem. 218: 1-13.

The present invention provides a fusion protein comprising two green fluorescent proteins (GFP) linked by a peptide containing at least one caspase degradation site, and an activator or inhibitor of caspase activity either in cells or in vitro. It relates to the use of fusion proteins in a method of identifying substances that are agents. The fusion protein comprises a donor GFP and an acceptor GFP. When the linker between the donor GFP and the acceptor GFP is perfect, the donor GFP is excited when the donor GFP is excited.
And FRET occurs between the acceptor and GFP. Cleavage of the linker by caspases separates the donor and acceptor GFPs, thus eliminating FRET between them.

The fusion protein is preferably derived from GFP which has altered and enhanced spectral properties compared to wild type GFP. Such GFPs are known to those of skill in the art (eg, US Pat. No. 5,625,048, International Patent Publication WO 97/2).
8261, International Patent Publication WO 96/23810). Particularly preferred is Aurora Biosciences Corp.
) (San Diego, CA) commercially available GFP, W1B, and TO
It is PAZ. W1B is F64L, S65T, Y66W, N146I, M15
3T and V163A contain changes from the wild-type GFP sequence (Ts
ien, 1998, Ann. Rev. Biochem. 67: 509-544,
(See page 1, page 519). TOPAZ is S65G, V68L, S72A, and T
It contains a change from the wild type GFP sequence, 203Y (Tsien, 19
98, Ann. Rev. Biochem. 67: 509-544, p. 519, see Table 1). The wild-type nucleotide and amino acid sequences of GFP are shown in FIG. 1 of International Patent Publication WO97 / 28261 and SEQ ID NO: 1, Tsien, 1998, Ann.
． Rev. Biochem. 67: 509-544, Figure 1, Prasher et al.,
1992, Gene 111: 229-233, and Figures 7A-B of the present application.

A wide variety of GFPs are suitable for use in the present invention, and certain general considerations should guide the selection of GFPs for use in fusion proteins. The excitation spectra of the donor and acceptor GFP should overlap as little as possible. This is because without directly exciting the acceptor
Allows the donor to be excited. The emission spectrum of the donor GFP should overlap as much as possible with the excitation spectrum of the acceptor GFP. The emission spectra of donor and acceptor should overlap as little as possible. The quantum yield of the donor and the absorption coefficient of the acceptor should be high enough.

Either the donor GFP or the acceptor GFP may be present in the amino-terminal or carboxy-terminal part of the fusion protein. Also, additional peptide sequences may be present at the amino-terminal or carboxy-terminal portion of the fusion protein. In other words, a suitable fusion protein of the invention may be abbreviated as follows.

NH ₂ -dGFP-linker-aGFP-COOH NH ₂ -aGFP-linker-dGFP-COOH NH ₂ -X-dGFP-linker-aGFP-Y-COOH NH ₂ -X-aGFP-linker-dGFP-Y- COOH where dGFP = donor GFP; aGFP = acceptor GFP; X = any amino acid or amino acid sequence; Y = any amino acid or amino acid sequence; X and Y
May be the same or different.

The complete fusion protein preferably has at least 1 ratiometric analysis.
． FRET to the extent that it will exhibit a FRET ratio of 5, preferably at least 2.0
Have the ability. That is, the ratio of the fluorescence emitted by the acceptor GFP divided by the fluorescence emitted by the donor GFP when the donor GFP is excited will be at least 1.5, preferably at least 2.0. Such a ratio allows sensitive detection of caspase activation and allows the advantages of ratiometric analysis as described herein to be exploited, thus rendering the method of the invention as a tool for high throughput screening. It is particularly suitable when used.

The linker is a peptide about 4 to 50 amino acids long, ie a short stretch of amino acids. Preferred are linkers less than 10 amino acids in length that contain a caspase cleavage site. The linker contains a single caspase cleavage site or multiple caspase cleavage sites that may be the same or different. Generally, the caspase degradation site is chosen to be recognized by a single caspase. However, for some purposes it may be preferable to use degradation sites recognized by multiple caspases. It may be further advantageous to use a linker containing multiple caspase cleavage sites recognized by different caspases. Such linkers are useful when it is desired to assay multiple caspase activators or inhibitors.

Analysis of caspase activity in whole cells generally involves detection of products generated by DNA fragmentation, membrane remodeling, chromatin aggregation, cell atrophy, alterations in mitochondrial membrane potential, and degradation of caspase substrates, such as: It has been limited to the determination of the ultimate biochemical and morphological markers of apoptosis. Such markers are downstream effects that are not measured in real time (ie, when caspase degradation is actually taking place), generally require collection of cells, and special reagents (eg antibodies to caspase substrates). Has the drawback that it is often required.

Methods of identifying activators and inhibitors of caspases in viable cells are provided by the present invention. These methods generally involve the use of cells engineered to express a fusion protein comprising two green fluorescent proteins (GFP) linked by a peptide containing at least one caspase degradation site. Generally, such cells are made by transfecting the cells with an expression vector containing DNA encoding the fusion protein. Fluorescence resonance energy transfer (FR) from donor GFP to acceptor GFP within a fusion protein in engineered cells
The amount of ET) is determined. Preferably, this determination is performed by ratiometric analysis. In a method of identifying activators of caspases, engineered cells are exposed to substances suspected to be activators of caspases. If the substance is in fact an activator, this results in an increase in intracellular caspase activity,
Causes degradation of the fusion protein linker. The two GFPs that make up the fusion protein then become free to move separately, and the FRs between them are
The amount of ET decreases. This decrease in FRET is preferably measured by ratiometric analysis. Methods for identifying inhibitors of caspases include caspase activity and / or
Or, except that the cells are treated to induce apoptosis and expose them to substances that are suspected to be inhibitors of caspases. If the substance is in fact an inhibitor, the degradation of the fusion protein will be reduced or eliminated, causing a reduction in FRET that is less than that seen when cells are not exposed to the substance. .

Accordingly, the present invention provides: (a) providing a cell containing a fusion protein containing a donor green fluorescent protein (GFP) and an acceptor GFP linked by a peptide containing at least one caspase cleavage site; (b) ) Measuring the amount of fluorescence resonance energy transfer (FRET) in the cell in the absence of a substance suspected to be an activator of caspase expression; (c) being an activator of caspase expression Exposing the cell to a suspected substance; (d) measuring the amount of FRET in the cell in the presence of the suspected activator of caspase expression: Including methods to identify. Here, a substance is a caspase activator if the amount of FRET measured in step (b) is greater than the amount of FRET measured in step (d).

The present invention provides: (a) preparing a cell containing a fusion protein containing a donor green fluorescent protein (GFP) and an acceptor GFP linked by a peptide containing at least one caspase cleavage site; (b) caspase (C) measuring the amount of fluorescence resonance energy transfer (FRET) in the treated cells in the absence of a substance suspected to be a caspase inhibitor; (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the treated cells; d) exposing the treated cells to a substance suspected of being a caspase inhibitor; (e) measuring the amount of FRET in treated cells exposed to a substance suspected of being a caspase inhibitor. What to do: Also included is a method of identifying a caspase inhibitor that comprises: Here, a substance is a caspase inhibitor if the amount of FRET measured in step (e) is greater than the amount of FRET measured in step (c).

In a particular embodiment of the above method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells or yeast cells. In another embodiment, the cells are L cells LM (TK ⁻ ) (ATCC CCL 1.3).
, L cells LM (ATCC CCL 1.2), HEK293 (ATCC CR
L 1573), Raji (ATCC CCL 86), CV-1 (ATCC
CCL 70), COS-1 (ATCC CRL 1650), COS-7 (A
TCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T
3 (ATCC CCL 92), NIH / 3T3 (ATCC CRL 1658)
), Hela (ATCC CCL 2), C127I (ATCC CRL 16)
16), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC C
CL 171), PC12 cells, C. elegans cells,
Zebrafish cells, Xenopus oocytes, Xenopus melanophores, or Saccharomyces cerevisiae
Y. cerevisiae) cells.

In a particular embodiment of said method, the cells are cells within a whole living organism such as eg C. elegans or zebrafish. The fusion protein is introduced into the organism by methods known to those skilled in the art. The advantage of using C. elegans and zebrafish is that these organisms are translucent and FRET can be observed in living animals. In this way, the activity of caspase activators and inhibitors can be monitored in real time in whole organisms. Alternatively, cells containing the fusion protein may be prepared from these organisms and such cells used to practice the methods of the invention.

In a particular embodiment, the invention provides that at least some cells express a fusion protein comprising a donor green fluorescent protein (GFP) and an acceptor GFP linked by a peptide comprising at least one caspase degradation site. Transgenic animals, such as transgenic mice, are used. Such transgenic animals are useful in understanding the role of caspases in conditions such as cardiovascular disease, neurodegenerative diseases, traumatic or septic shock, and stroke. For example, transgenic mice expressing the fusion protein in neural tissue are useful models for assessing the effects of caspase inhibitors on chronic or acute injury, such as neurodegenerative disease, brain injury, or stroke. If this model is applied to seizures, it will induce seizures in transgenic mice in the presence or absence of caspase inhibitors. Then
To determine whether caspase inhibitors reach specific areas of the brain and inhibit caspases in those areas, mouse brain tissue will be dissected and imaged microscopically. By analysis of the effect of inhibitors combined with analysis of seizure injury,
It can be determined whether there is a correlation between caspase inhibition and protection from stroke injury. Thus, the present invention may be used to assess the ability of caspase inhibitors to protect brain tissue from stroke injury.

In certain embodiments of the above methods, the cells are adherent cells rather than suspension cells. In certain embodiments, the method is performed in the wells of a microtiter-like plate and the cells are not collected or detached from the wells. That is, the cells remain attached to the bottom of the well throughout the practice of the method.

In certain embodiments of the above methods, an expression vector encoding the fusion protein is transfected into cells. Such expression vectors are described, for example, in Sambrook, Fritsch, and Maniatis, 1989, Mol.
Equal Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Pre
ss, or PCR Primer, A Laboratory Manual
, C. W. Dieffenbach and G.W. S. Dveksler, 1995
, Cold Spring Harbor Laboratory Press
Can be made by well-known recombinant DNA methods such as those described in. One method would involve starting from standard expression vectors. The DNA sequences encoding the donor GFP and the acceptor GFP are cloned into the polylinker site of a standard expression vector, preferably after PCR of GFP so that the sequences are flanked by restriction enzyme cleavage sites. After cloning the GFP into an expression vector, there will be one or more restriction sites between the GFP sequences that allow the linker to be cloned. The linker sequence is chosen such that the donor GFP, acceptor GFP, and linker will all be in the same reading frame in the final expression construct. Two GFs separated by a 20 amino acid linker with a protease site (in this case the Factor Xa site)
Examples of construction of fusion proteins containing P are described in Mitra et al., 1996, Gene 1
73: 13-17. 40 of International Patent Publication WO 97/28261
Example 1, starting on page, discloses a method for constructing GFP fusion proteins containing linkers with diverse protease cleavage sites. The general procedure disclosed in these references can be used as a guide for constructing the caspase-specific fusion proteins of the invention.

Transfection can be accomplished by any method known to those of skill in the art, such as calcium phosphate or calcium chloride mediated transfection, electroporation, infection with retroviral vectors. The cells can be transiently or stably transfected.

The present invention provides a DN encoding a fusion protein containing two green fluorescent proteins (GFP) linked by a peptide containing at least one caspase cleavage site.
Including A. The present invention also includes an expression vector containing a DNA encoding a fusion protein containing two green fluorescent proteins (GFP) linked by a peptide containing at least one caspase cleavage site, and a host cell containing these expression vectors. .

A wide variety of standard expression vectors can be used to produce expression vectors that encode fusion proteins. Suitable commercially available expression vectors include, but are not limited to, pMC1neo (Stratagene, La Jolla, CA.
), PSG5 (Stratagene, La Jolla, CA), pcDNA
I and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (
Invitrogen, Palo Alto, CA), EBO-pSV2-ne.
o (ATCC 37593), pBPV-1 (8-2) (ATCC 37110)
), PdBPV-MMTneo (342-12) (ATCC 37224), p.
RSVgpt (ATCC 37199), pRSVneo (ATCC 3719)
8), pSV2-dhfr (ATCC 37146), and pIRES1neo.
(Clontech, Palo Alto, CA). The choice of vector will depend on the cell type used, the level of expression desired, etc. When expressing GFP in mammalian cells, it may be advantageous to construct a form of GFP with modified codons that follow those codons preferred for mammalian cells (Zolotukhin et al., J. Virol. 1996, 70). : 4646
-46754; Yang et al., 1996, Nucl. Acids Res. 24:
4592-4593). Another way to improve GFP expression in mammalian cells is to use an additional codon immediately after the initiation methionine to provide an optimal ribosome binding site (Crameri et al., 1996, Na.
pure Biotechnology 14: 315-319).

For certain embodiments, the GFP fusion protein may be cloned under the control of an inducible promoter, such as a promoter induced by dexamethasone, metallothionein, or related analogs such as tetracycline and doxycycline. It would be advantageous to use a vector. This allows
The toxicity problems that can result from uncontrolled high level expression of GFP can be avoided. A suitable vector containing an inducible promoter is Clontech (C
lontech) (Palo Alto, CA) pTRE vector. This vector contains a tetracycline inducible promoter. Other such vectors are well known in the art and many are commercially available.

In certain embodiments, the donor GFP and the acceptor GFP are W1B, F64L change, S65T change, Y66W change, N146I change, M1.
Wild-type GFP having 53T and V163A changes (SEQ ID NO: 6); TOPAZ, S65G change, V68L change, S72A change, and T203Y change wild-type GFP (SEQ ID NO: 6); Wild-type GFP (SEQ ID NO: 6); Wild type GFP with altered Q80R (SEQ ID NO: 6) (Chalpie et al., 19
94, Science 263: 802-805); wild type GFP with S65T change (SEQ ID NO: 6); wild type GFP with Y66 change W (SEQ ID NO: 6); wild type GFP with Y66 change F (SEQ ID NO: 6). Wild type GFP having F64L change, S65T change, and V163A change (SEQ ID NO: 6); Wild type GFP having S65G change, S72A change, and T203F change (SEQ ID NO: 6); having change S65G, S72A, and T203H Wild type GFP (SEQ ID NO: 6
); Wild-type GFP having Cycle3, F99S change, M153T change, and V163A change (SEQ ID NO: 6); EGFP, wild-type GFP having F64L change and S65T change (SEQ ID NO: 6)
); Emerald, S65T change, S72A change, N149K change, M153T
Wild type GFP with changes and I167T changes (SEQ ID NO: 6); Wild type GFP with H9, S202F changes and T203I changes (SEQ ID NO: 6).
); Wild-type GFP having H9-40, T203I change, S72A change, and Y145F change (SEQ ID NO: 6); 10C Q69K, S65G change, V68L change, Q69K change, S72A change, and T203Y change wild-type GFP (SEQ ID NO: 6); SEQ ID NO: 6); 10C, wild type GFP with S65G change, V68L change, S72A change, and T203Y change (SEQ ID NO: 6); W7, Y66W change, N146I change, M153T change, and V163A change with wild type GFP (SEQ ID NO: 6); SEQ ID NO: 6); W1C, S65A change, Y66W change, S72A change, N146I change, M1
Wild type GFP with 53T change and V163A change (SEQ ID NO: 6); BFP, wild type GFP with Y66H change (SEQ ID NO: 6); P4-3, wild type GFP with Y66H change and Y145F change (SEQ ID NO: 6) ); And wild type GF with EBFP, F64L change, Y66H change, and Y145F
P (SEQ ID NO: 6): selected from the group consisting of:

A wide variety of suitable donor and acceptor GFPs and their spectral characteristics are described in Tsien, 1998, Annu. Rev. Biochem. 67:
509-544, pages 519, Table 1.

In some embodiments of the method of identifying an activator or inhibitor of caspase, the step of treating the cell to activate caspase comprises viral infection; growth factor depletion; anticancer agent; UV exposure; stauros. Porin, actinomycin D, etoposide, glucocorticoid, camptothecin, thapsigargin (thaps
igargin), cyclohexamide, rapamycin, ceramide, glutamate, N-methyl-D-aspartic acid, kainic acid, lectin, tributyltin, vincristine, vinblastine, cisplatin, pentoxifylline, cytotrienin A, 6- Hydroxydopamine, 1,
Treatment with 10-phenanthroline, nitric oxide, or sodium butyrate; forced c-myc expression; or stimulation of members of the tumor necrosis factor receptor (TNFR) family (eg, Fas / Fas ligand interaction) ( Ashkenazi & Dixit, 1998, Science 28
1: 1305-1308) :. These methods are merely exemplary and many other suitable methods are known to those skilled in the art.

In certain embodiments, the step of measuring the amount of FRET is performed by ratiometric analysis. In certain embodiments, measuring the amount of FRET is performed by measuring an increase or decrease in the release of donor GFP. In certain embodiments, measuring the amount of FRET is performed by measuring an increase or decrease in the release of acceptor GFP.

The conditions under which the step of exposing the cells to the substance is carried out are those typically used in the art for studying protein-ligand interactions or exposing cells to compounds, such as physiological pH. , Salt conditions of commonly used buffers or tissue culture media such as PBS, temperatures of about 4-55 ° C. In certain embodiments, it may be advantageous to include a substance that quenches background fluorescence in the tissue culture medium in which the cells are grown.

In certain embodiments, the method is performed as a method of high throughput screening. In a particular embodiment, the method is repeated at least 5,000 times, preferably at least 50,000 times, more preferably at least 100,000 times in 24 hours. In a particular embodiment, at least 5,000, preferably at least 50,000, more preferably at least 100,000 different substances are tested in 24 hours. In certain embodiments, the method is performed in a microtiter-like plate. In certain embodiments, the microtiter-like plates are 96, 384, 1,536.
There are 3,064, 3,456, or 9600 wells.

In a particular embodiment of the method of identifying a caspase activator or caspase inhibitor, step (c) does not involve transfecting the cell with a gene that induces apoptosis or caspase activation, and The measuring step does not include FACS analysis or flow cytometry.

In particular embodiments, the caspase is a mammalian caspase. In certain embodiments, the caspase is human caspase. In certain embodiments, the caspase is caspase-1 (also known as interleukin-1β converting enzyme (ICE))
(Thornberry et al., 1992, Nature 356: 768-774.
Cerrretti et al., 1992, Science 256: 97-100).
Caspase-2 (also known as ICH-1) (Wang et al., 1994, Cel
78: 739-750); Caspase-3 (also known as apopain, CPP32, Yama) (Nicholson et al., 1995, Nature 376: 37-).
43; Rotonda et al., 1996, Nat. Struct. Biol. 3: 6
19-625); Caspase-4 (also known as ICE _rel- II, TX, ICH-2) (M
unday et al., 1995, J. Am. Biol. Chem. 270: 15870-15
876; Kamens et al., 1995, J. Am. Biol. Chem. 270: 152
50-15256; Facheu et al., 1995 EMBO J. et al. 14: 1914
-1922); Caspase-5 (ICE _rel- III, also known as TY) (Munday
Et al., 1995, J. Am. Biol. Chem. 270: 15870-15876; F
aucheu et al., 1996, Eur. J. Biochem. 236: 207-2
13); Caspase-6 (also known as Mch2) (Fernandes-Alnem)
ri et al., 1995, Cancer Res. 55: 2737-2742); Caspase-7 (also known as Mch3, ICE-LAP3, CMH-1) (
Fernandes-Alnemri et al., 1995, Cancer Res. 5
5: 6045-6052); Caspase-8 (also known as MACH, FLICE, Mch5) (Bold
in et al., 1996, Cell 85: 803-815; Muzio et al., 199.
6, Cell 85: 817-827); Caspase-9 (ICE-LAP6, also known as Mch6) (Duan et al.,
1996, J. Am. Biol. Chem. 271: 16720-16724); Caspase-10 (also known as Mch4); Caspase-11; and Caspase-12 :.

In a particular embodiment, the caspase degradation site is DXXD (SEQ ID NO: 7), where X is any naturally occurring amino acid; DEXD (SEQ ID NO: 8), where X is any DEVD (SEQ ID NO: 9); DEHD (SEQ ID NO: 10); DETD (SEQ ID NO: 11); DGPD (SEQ ID NO: 12); DEPD (SEQ ID NO: 13); DELD (SEQ ID NO: 14) ); DAVD (SEQ ID NO: 15); DMQD (SEQ ID NO: 16); DSID (SEQ ID NO: 17); DVPD (SEQ ID NO: 18); DQTD (SEQ ID NO: 19); DSLD (SEQ ID NO: 20); WEHD (SEQ ID NO: 21) ZEXD (SEQ ID NO: 22), where X is any naturally occurring amino acid and Z is either I, L or V; LE D (SEQ ID NO: 23); VEHD (SEQ ID NO: 24); LETD (SEQ ID NO: 25); YVAD (SEQ ID NO: 26); LVAD (SEQ ID NO: 27); ELPD (SEQ ID NO: 28); SRVD (SEQ ID NO: 29); VEID (SEQ ID NO: 30); and IETD (SEQ ID NO: 31) :.

Thornberry et al., 1997, J. Am. Biol. Chem. 272: 17
907-17911 discloses degradation site preferences for various caspases. This publication describes a method for identifying caspase degradation sites by using a positional scanning combinatorial library. In general, suitable caspase degradation sites for use in the present invention are known to those of skill in the art to be any sequence determined by such methods to be a caspase degradation site, or a caspase degradation site. Contains any array.

In certain embodiments of the above methods, the caspase for which the activator or inhibitor is identified is a caspase that is naturally expressed in cells. In other embodiments, the caspase for which an activator or inhibitor is identified is provided by transfecting a cell with an expression vector that directs expression of the caspase. This can be done to provide for expression of the caspase in cells in which the caspase is not naturally expressed (eg yeast or C. elegans which do not have endogenous caspase), or where the caspase is naturally expressed at low levels. Can be performed to provide high levels of caspase expression in living cells. In a variation of this procedure, the caspases that are transfected into the cells are those that contain a mutation or modification of the amino acid sequence as compared to the wild type caspase. Another one
In one variation, the caspase is from a species other than the cell from which it was derived.

Although the method is explicitly concerned with testing whether a “one” substance is an activator or inhibitor of caspases, such a method may be used to aggregate a substance, eg a combinatorial library. It will be apparent to those skilled in the art that they are suitable for testing aggregates of natural products, etc. to determine whether members of such aggregates are activators or inhibitors of caspases. Let's do it. Thus, the use of an aggregate of substances, or individual members of such an aggregate, as substances in the method is included within the scope of the invention.

After the substance has been identified as an activator or inhibitor of caspases by the above method, whether or not the substance acts at the level of caspase proteolysis rather than at the level of upstream events that activate caspases is determined. It may be desirable to decide. This can be done, for example, by setting up an in vitro assay in which purified caspase is incubated with the purified fusion protein. The absence of other components of the apoptotic pathway in such in vitro assays (FRET
It means that any effect on the caspase activity evoked by the substance (as measured by the change in) would be due to the direct effect on the caspase itself. Examples of such in vitro assays are described in Examples 2 and 9 herein and FIG.

Another method for determining the level at which a caspase inhibitor acts is step (b) of this method (treatment of cells to activate caspase) and step (b).
d) (exposure of cells to substances suspected to be caspase inhibitors). This time will allow active caspases to form. This active caspase will result in the degradation of large amounts of the fusion protein and will therefore result in little FRET. If the substance is an inhibitor, exposure of cells to the substance will increase the amount of FRET. If the inhibitor acts at the level of caspase proteolysis, this increase should occur rapidly, with about the same amount of time it takes for the inhibitor to enter the cell. If the inhibitor acts at an upstream event, eg acts to pause an upstream caspase activation event, the effect of the inhibitor on caspase proteolysis should occur at a relatively slow rate. In such cases, the increase in FRET should also occur at a relatively slow rate.

The amount of time that the treatment of step (b) actually takes to activate the caspase depends on the cell type used in the method, the nature of the treatment (eg, the compound used to activate the caspase). Species), and the concentration of compound used to activate the caspases, and will depend on a number of factors. Some compounds can activate caspases and / or induce apoptosis very rapidly (ie, in minutes), and some require incubation for hours, or even overnight. In most cases, if it is desired to elucidate the level at which the substance acts, the treatment that activates the caspase is treated about 2-8 hours before exposing the cells to the substance suspected to be a caspase inhibitor. Should be implemented. However, in some embodiments, the cells may be exposed to the substance at the same time as, or prior to, the application of the treatment that activates caspases. This may depend on factors such as the metabolic stability of the substance, cell permeability, or even the kinetic characteristics of the substance / caspase interaction, eg rate, affinity, K _i . A person skilled in the art will readily be able to determine the appropriate relationship between treatment and material exposure, depending on the particular details of the method being performed.

The measurement of the amount of FRET is generally carried out by exciting the donor GFP by using a lamp, laser or other light source tuned to the excitation wavelength of the donor GFP. The excitation energy absorbed by the donor is emitted by the donor at its emission wavelength, or the excitation energy is at least partially transferred to the acceptor GFP, which in turn emits excitation energy at its emission wavelength. To do. By measuring the amount of emission at both the acceptor and donor wavelengths, it is possible to determine how much FRET has occurred. A high acceptor / donor emission ratio means a large amount of FRET, and conversely, a low acceptor / donor emission ratio means a small amount of FRET.

The magnitude of the donor-acceptor emission ratio is unaffected by such factors as the absolute amount of fusion protein or its degradation products, the optical density of the sample being assayed, the brightness of the excitation source, or the sensitivity of the detector. , The use of ratiometric analysis is preferred as it is a measure of the degradation of the fusion protein.

The use of ratiometric analysis is also preferred because ratiometric analysis provides a level of sensitivity suitable for use in high throughput screening methods.

The sensitivity of the method of the present invention is also increased by using super bright GFP as donor GFP. The conventional technique is not a super bright GFP but a blue fluorescent protein (
A type of GFP known as BFP) was used as the donor GFP (Xu et al.,
1998, Nucl. Acids Res. 26: 2034-2035 (Xu)
). BFP has a mutation at position 66 that changes wild-type tyrosine to histidine. This Y66H change results in proteins with relatively weak fluorescence (ie, fluorescence quantum yield of less than about 0.30). Such weak fluorescence may be suitable for flow cytometry (eg, FACS analysis such as Xu) but not for ratiometric analysis and therefore suitable for use in high throughput screening. Absent. The fusion protein described in Xu is potentially useful in high throughput screening methods to identify caspase inhibitors performed in microtiter-like plates, which is a preferred embodiment of the invention, and may provide a reasonably sensitive readout. Extremely low. From a careful analysis of Xu,
It is clear that Xu does not disclose a method for identifying caspase inhibitors using FRET. The only way Xu used a caspase inhibitor involved the use of Western blotting to identify fragments of the fusion protein of Xu. In the presence of the caspase inhibitor, Xu was able to demonstrate the disappearance of fragment generation (see page 2035, Figure 1B). Where Xu used fluorescence measurements (203
Page 5, FIG. 2) was limited to measuring the effect of activation rather than inhibition of caspases.

The difficulty of using BFP for ratiometric analysis is especially pronounced in viable cells. For a discussion of this issue, see Pollock & Heim.
, 1999, Trends Cell Biol. 9: 57-60, page 58. In view of this difficulty, a preferred embodiment of the present invention uses a donor GFP that does not have a histidine at position 66, ie a GFP that does not have a Y66H change.

As understood herein, high throughput screening requires sample throughputs that exceed the current capabilities of methods such as flow cytometry. Flow cytometry requires steps such as cell collection, cell washing, loading cells into a suitable device, analyzing cells one at a time. High throughput screening is preferably performed in multi-well microtiter-like plates rather than flow cytometry equipment. Multi-well plates allow parallel analysis of thousands of assays every few minutes, whereas flow cytometry only allows analysis of hundreds of assays per minute (Nolan et al.,
1999, Drug Discovery Today 4: 173-180,
See page 179, right column). A wide variety of automated analyzers suitable for performing high throughput screening are known that can be used with microtiter-like plates. For example, US Pat. No. 5,670,113, US Pat.
See 39,744, and U.S. Pat. No. 4,626,684.

The method of the present invention is particularly suitable for high throughput screening as it exhibits several advantages over the prior art. The use of ratiometric analysis with super bright GFP obviates the tedious and time consuming step of collecting cells from multi-well plates, as required in flow cytometric methods such as FACS analysis. To do. Therefore, the method of the present invention can be applied to Hela cells, 2
It is particularly suitable for use in adherent cells such as 93 cells, L cells, CHO cells, PC12 cells, COS cells, or 3T3 cells. The method described herein avoids the need for tedious steps such as collecting adherent cells from multi-well plates, Xu et al., 1998, Nucl. Acids Res. 26:20
It provides a means to screen far more compounds than can be performed using less efficient flow cytometric methods, such as those described in 34-2035. Even when using the method of the invention with suspended cells, the method is
It has the advantage over prior art methods that it is not necessary to collect suspended cells prior to assaying for FRET.

The prior art used an 18 amino acid linker region within a fusion protein containing a blue fluorescent protein and a green fluorescent protein with a caspase cleavage site (Xu
Et al., 1998, Nucl. Acids Res. 26: 2034-2035 (X
u)). Xu considered that the use of such a long linker region was necessary "to render this region soluble and susceptible to degradation by CPP32 (p. 2034, right column, top)". Contrary to the teachings of the prior art, we have found that relatively short linker regions (eg,
(10, 9, 8, 7, 7, 6, 5, or 4 amino acids) was found to be more efficient. See FIG. 1B, where the linker region of fusion protein WT-1x is shown to be only 9 amino acids long. Contrary to the expectations based on the prior art, the present inventors have shown that such a short linker allows caspase degradation and efficient FRE.
It was found to be sufficient to allow the T measurement. Data for WT-1x show that caspase degradation of WT-1x can be readily detected in viable cells using ratiometric analysis, see FIG. 4B. A comparison of FIGS. 4B and 4A shows that our discovery of the use of such ratiometric analysis associated with short linkers is valuable. Western blot analysis of Hela cells expressing WT-1x induced to undergo apoptosis showed only trace amounts of WT-1x degradation products that could be easily overlooked, whereas ratiometric analysis of the cells showed degradation. Clearly indicates that (reduction of FRET ratio by more than 50%) has occurred. Thus, the use of ratiometric analysis gives the assay for caspase activity much higher sensitivity than the use of Western blotting. Of course, such increased sensitivity is of great value in the use of such assays to identify activators of caspase expression and inhibitors of caspase activity.

The present invention provides fusion proteins in which all amino acids except the 4 or 5 amino acid linker, which is the caspase cleavage site, are derived from the amino acids of the donor and acceptor GFP, and the methods described herein. Including its use in. In some embodiments, it may be advantageous to use glycine or another amino acid with a small chain for the critical side (ie, immediately after the aspartate cleavage position) to avoid steric hindrance of the caspases. .

Inhibitors of caspases identified by the methods of the invention will have a variety of uses. Such inhibitors would be useful in treating conditions where it would be beneficial to reduce the level of caspase activity or apoptosis. Such conditions include, for example, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; traumatic brain injury; stroke; ischemia-reperfusion injury; graft-versus-host disease; and autoimmune disease. It will be apparent to those skilled in the pharmaceutical arts that caspase inhibitors will have useful pharmacological activity. Caspase-1 (interleukin-1β converting enzyme)
Inhibitors are currently in clinical trials for the treatment of inflammatory diseases (Press r
Release, Vertex Pharmaceuticals, Inc. , 1
October 29, 998). Caspase inhibitors have been shown to reduce neuronal injury in a rabbit bacterial meningitis model (Braun et al., 1999).
, Nature Medicine 5: 298-302).

The activators of caspases identified by the methods of the present invention are conditions where it is beneficial to increase the level of caspase activity or apoptosis, such as cancer, or any disease involving hyperplastic or neoplastic transformation. , Is likely to be useful in the treatment of graft-versus-host disease. Caspase activators / apoptosis inducers are in clinical trials for use against graft-versus-host disease (Press releases).
e, Ariad Pharmaceuticals, May 6, 1999).

The present invention includes a pharmaceutical composition comprising an activator and an inhibitor of caspase identified by the above method. Activators and inhibitors are generally combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition. Examples of such carriers, and methods of formulating pharmaceutical compositions containing activators and inhibitors and carriers, are described in Remington.
Found in on's Pharmaceutical Sciences. Such compositions will contain therapeutically effective amounts of activators and inhibitors to form pharmaceutically acceptable compositions suitable for efficient administration.

The therapeutic or prophylactic composition is administered to an individual in an amount sufficient to treat or prevent a condition in which caspase activity is abnormal. The effective amount can vary depending on a variety of factors such as the individual's condition, weight, sex, and age. Other factors include the mode of administration. Appropriate amounts can be determined by the skilled physician.

The composition may be used alone at a suitable dose. Alternatively, simultaneous or sequential administration of other agents may be desirable.

The compositions can be administered in a wide variety of therapeutic dosage forms contained in conventional vehicles for administration. For example, in oral dosage forms such as tablets, capsules (including sustained-release and sustained-release formulations, respectively), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups, and emulsions. Or, the composition may be administered by injection. Similarly, they are administered intravenously (using all forms well known to those skilled in the pharmaceutical arts).
Both bolus and infusion), intraperitoneal, subcutaneous, topical (with or without occlusion), or intramuscular.

Advantageously, the composition may be administered in a once daily dose, or the total daily dose is 1
It may be administered in divided doses of two, three or four times daily. Further, the composition is
It may be administered via topical use of suitable intranasal vehicles, in intranasal form or via transdermal routes, using the forms of transdermal skin patches well known to those of ordinary skill in that art. Dosing administered in the form of transdermal delivery systems will, of course, be continuous rather than intermittent throughout the dosing schedule.

Dosage regimens utilizing the composition may include patient type, species, age, weight, sex, and medical condition; severity of the condition to be treated; route of administration; patient kidney, liver, and cardiovascular function. As well as selected according to a variety of factors, including the particular composition used. A physician of ordinary skill can readily determine and prescribe the effective amount of the composition required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of the composition within the range that provides efficacy without toxicity requires a regimen based on the kinetics of the composition's reachability to target sites. This includes consideration of composition distribution, equilibration, and removal.

[0079]   The following non-limiting examples are presented to further illustrate the present invention.

Example 1 Fusion Protein Containing a Group II Caspase Degradation Site An induction composed of tandem green fluorescent protein (GFP) molecules linked by an amino acid sequence that is recognized and degraded by a Group II caspase (eg, caspase-3). A recombinant caspase substrate was engineered to encode a possible fusion protein (FIG. 1B) (Thornberry et al., 1997, J. Biol. Ch.
em. 272: 17907-17911). Nonspecific G due to long-term overexpression
To avoid potential problems with FP-mediated toxicity, a tetracycline inducible expression system was used (Gossen et al., 1994, Curr. Opin. Biote.
chnol. 5: 516-520). The caspase recognition motif (DEVD (SEQ ID NO: 9)) embedded within these fusion proteins is a well-characterized substrate for caspase-3 in apoptotic cells, poly- (ADP-ribose) -polymerase (PARP). Derived from the protease degradation site identified in (Lazebnik et al., 1994, Nature 371: 346-34.
7). In addition, based on studies investigating the degradation of combinatorial fluorogenic tetrapeptide substrate libraries, this site was defined as the optimal group II caspase degradation motif (Thornberry et al., 1997, J. Biol. Che.
m. 272: 17907-17911).

Mutant GFP protein (W1B, To used to generate the fusion)
paz; Aurora Biosciences, San Diego, CA)
The spectral features (excitation and emission) of the are well suited to distinguish between degraded and undegraded forms of fusion proteins using standard fluorometric assay (Tsien, 1998,
Annu. Rev. Biochem. 67: 509-544). W1B has double excitation (434/452 nm) and emission (476/505 nm) peaks, and Topaz has excitation and emission maxima of 514 nm and 527 nm, respectively. In principle, caspase-3 mediated degradation of these substrates should prevent fluorescence resonance energy transfer (FRET) between the linked GFP molecules by physical separation of the molecules. Excitation wavelength (ex:
Excitation at 434 nm) causes excitation fluorescence (Topaz, em: 527 nm) emitted by the acceptor molecule and fluorescence emitted by the donor molecule (W
The degradation of the substrate can be quantified as a ratio divided by 1B, em: 476 nm). In the cell-based assay described herein, an increase in the ratio of emitted fluorescence (527/476) at these wavelengths would reflect inhibition of caspase activity.

Various criteria must be met for successful development of this assay. For example, the caspase-3 degradation motif embedded in the linker region
It was necessary to demonstrate that it could be efficiently degraded by caspase-3 without interfering with FRET between tethered molecules. To this end, disassembly and FRET
To optimize both efficiency, several potential linker regions were designed containing single or tandem DEVD (SEQ ID NO: 9) sites. The construct, designated WT-2xL, flanks a tandem DEVD (SEQ ID NO: 9) site in a hinge region rich in glycine and serine residues that may impart flexibility to the GFP fusion protein and promote energy transfer. (Hall, 1998, J. Pare).
inter Internal. Nutr. 22: 393-398; Freund et al., 1993, FEBS Lett. 320: 97-100; Steinert et al., 1991, Int. J. Biol. Macromol. 13: 130-139
). 2A-B show transfected He overexpressing the fusion protein.
It shows a direct correlation between the investigated linker region length and FRET efficiency in la cells. A corresponding decrease in FRET efficiency was observed as the length of the linker separating the GFP molecules increased. Energy transfer is tandem DE
Single DEV rather than a fusion containing the VD (SEQ ID NO: 9) motif (WT-2x)
The fusion containing the D (SEQ ID NO: 9) motif (WT-1x) was more efficient. Interestingly, the inclusion of a flexible hinge region within the linker does not confer any particular advantage, but instead increases the FRET efficiency of the WT-2xL fusion protein compared to its parental form (WT-2x). It worked to weaken.

Example 2 In Vitro Expression and Degradation of a Fusion Protein Containing a Group II Caspase Degradation Site First, a [ ³⁵ S] -Met radiolabeled protein translated in wheat malt lysate was used to tandem GFP for degradation by caspases. The sensitivity of the substrate was investigated. Proteins were incubated in vitro with increasing concentrations of purified recombinant caspase-3 and degradation products were separated by polyacrylamide gel electrophoresis and quantified by autoradiographic analysis of the gel. Single DEVD (SEQ ID NO: 9) containing substrate (WT-1 ×) and dual DEVD (SEQ ID NO: 9) containing substrate (WT
Comparison with -2 ×), as estimated by the _K cat / _{K m} values, WT -
It was shown that 2x was degraded approximately 4-fold more efficiently (Figure 3). This result is consistent with the data using other recombinant caspase substrates artificially introduced with the artificial DEVD (SEQ ID NO: 9) motif (unpublished observation). This is DEVD
(SEQ ID NO: 9) By multiplexing the motif, an authentic group II caspase substrate (
For example, PARP, U1-70kDa, and DNA-PKcs) (Cascio)
la-Rosen et al., 1996, J. Am. Exp. Med. 183: 1957-19
It has also been proved that it is possible to generate an artificial substrate that is degraded as efficiently as 64). To compare the efficiency with which these fusion proteins can be degraded by caspases in whole cells, He transfected with various constructs was compared.
La cells were stimulated to undergo apoptosis and degradation products were analyzed by immunoblot analysis using a polyclonal anti-GFP antibody (Fig. 4A). It has been previously demonstrated by several studies that staurosporine induces proteolytic activation of caspase-3 in Hela cells. Increasing the number of DEVD (SEQ ID NO: 9) sites in the linker region separating the two GFP molecules has a negative impact on FRET efficiency, in contrast to the tandem DEVD (sequence No. 9) The fusion protein carrying the motif was degraded more efficiently after staurosporine treatment. Furthermore, cells transfected with the parental construct (WT) that did not contain the caspase-3 degradation site were unable to induce proteolysis of the fusion protein in response to apoptosis. In addition, staurosporine treated cultures were incubated with zVAD-fmk, an irreversible non-specific (pan) caspase peptide inhibitor, to disrupt degradation of DEVD (SEQ ID NO: 9) containing fusion proteins. Attenuated.
This data confirms that the recombinant GFP substrate can be used to monitor activation and inhibition of group II caspases in cells.

Example 3 Expression and Degradation of Fusion Proteins Comprising a Group II Caspase Degradation Site in Viable Cells To determine whether inhibition of caspase degradation could result in measurable and dose-dependent changes in FRET-WT- Caspase inhibition by zVAD-fmk was titrated in cultured Hela cells stably overexpressing the 1 × construct. This fusion construct was selected for analysis because it shows a reasonable compromise between energy transfer efficiency and degradability as a substrate (Figures 2A-B and Figure 3). Caspase inhibition was investigated using fully adherent cells seeded in a 96-well microtiter-like plate format. A fluorometer equipped with an appropriate filter set to detect FRET was calibrated to read the fluorescence emitted from whole cells present on the surface of the plate. After treatment with staurosporine, alone or in combination with different concentrations of inhibitor (zVAD-fmk), cells were examined by fluorescence analysis. The data in FIG. 5 show that treatment of WT-1 × expressing cells with increasing concentrations of zVAD-fmk increased WT-1 × derived FRET simultaneously and in a dose-dependent manner (IC ₅₀ = 23 μM). The dose response curve is similar to that obtained using other biochemical markers of apoptosis, including DNA fragmentation (
Not shown). These results demonstrate that quantification can be performed directly on cells without the need for further manipulation (eg cell collection, lysate preparation). Furthermore, the ability to perform the assay in a multi-well format underscores its utility as a screening tool for drug delivery.

Example 4 High Throughput Screen In addition, to examine the availability of the tandem GFP assay as a high throughput screening method to identify caspase inhibitors, a single compound or a mixture of 10 compounds per well. Duplicate sets of 96-well drug plates containing either of The final concentration of each drug in these plates is
It was 400-600 μM. Preliminary screening of these plates revealed the absence of caspase inhibitors in the test compound population (data not shown). Individual wells were supplemented with varying concentrations of a known caspase inhibitor (zVAD-fmk) to demonstrate that they would be detected if caspase inhibitors were present in these plates. Furthermore, in order to avoid bias in the interpretation of the data, zVAD-fmk can only be obtained after the experiment is completed.
The experiment was performed blindly so that the location of each well containing In these experiments, two wells of each plate contained three different concentrations of zVAD-
fmk (2.5 μM, 25 μM, and 250 μM) was randomly supplemented and each plate was assayed in duplicate. The results of the screening are shown in Figures 6A-B. 6A and 6B represent plate arrays containing single compounds and drug mixtures, respectively. Data investigations, regardless of plate format
We showed that duplicate wells containing either 25 μM or 250 μM zVAD-fmk were readily identified. As expected, zVAD-fmk could not be detected at concentrations (2.5 μM) that could not inhibit the degradation of tandem GFP substrate (see FIG. 5). In one of the plates containing each compound, a single well (coordinate G3) gave a positive signal not due to the presence of zVAD-fmk. However, this sample represented a false positive result because it was not registered as a hit in duplicate plates. Only one further false positive hit was detected in the same well position (coordinate G3) in the plate containing the drug mixture. This sample was scored as a positive hit in duplicate plates, but the fluorescence emission values recorded at wavelengths of both 490 nm or 530 nm far exceeded the maximum emission values obtained from control wells, which , Show that this false positive hit represents highly fluorescent compound (s). Finally, the rate of false positive hits is extremely low (0.1%
~ 1.0%), any false positive hits detected in this assay can be easily excluded by the use of a data analysis algorithm designed to flag such hits. is important.

Example 5 Construct The W1B GFP mutant was constructed in pRSETA-hW1B (Aurora Bios).
Sciences, San Diego, CA), EcoRI / W1B / Af
AII / AgeI fragment was amplified by polymerase chain reaction (PCR). To the initiation methionine of W1B Kozak consensus (gccgccaccat)
gg; SEQ ID NO: 32) was inserted and the stop codon was deleted (Kozak, 198).
4, Nucleic Acid Res. 12: 857-872). Topaz
Is pRSETA-TOPAZ (Aurora Biosciences, Sa
nDiego, CA) as an AgeI / NheI / TOPAZ / BamHI fragment by PCR. The starting methionine of Topaz was deleted. W
1B and Topaz for EcoRI / AgeI fragment and AgeI / Ba, respectively.
As an mHI fragment, pIRES1neo (Clontech, Palo Alt
o, CA) to the EcoRI / BamHI site and pIRES1n
eo-WT was generated. This created a tandem fusion of W1B and TOPAZ separated by a 6 amino acid linker encoded by AflII / AgeI / NheI sites. pIRES1neo-WT-1x, pIRES1
neo-WT-2x and pIRES1 neo-WT-2xL is the parent pIRES
It was generated by ligation of a synthetic oligonucleotide encoding a DEVD (SEQ ID NO: 9) linker sequence into the AflII / NheI sites of the 1neo-WT plasmid.

Example 6 Transfection Hela cells (7.5 × 10 ⁵ ) were passaged into a 60 mm tissue culture dish, and the next day,
Lipofectamine Plus reagent (Li
The plasmid DNA was transfected with 2.5 μg using fe Technologies. After allowing the cells to express the GFP fusion for a further 48 hours,
Analysis was carried out. To determine the maximum possible FRET efficiency, to inhibit the caspase activity produced as a result of transfection, zVAD-fmk (100 μM) (Enzyme Systems) was used during the transfection procedure.
Products) were included in the medium and maintained for 48 hours. Also in the Western blotting experiment, zVAD-fmk was added to the medium as described above. For the induction of apoptosis, after the first 48 hours of expression, staurosporine (1 μm
M) treated cells for 4 hours.

Example 7 Preparation of Lysate Hela cells were detached from 60 mm tissue culture dishes into growth medium and incubated at 4 ° C., 2,000.
Pellet at 0xg for 10 minutes. Wash the cell pellet with cold PBS, 1 ml,
Pelletized as above. TET buffer (50 mM Tris, pH 7.5 / 2
Cell pellets were disrupted using 100 microliters of mM EDTA / 1% Triton X-100) and the resulting lysates were grown at 4 ° C for 10Ox10 g.
Clarified by centrifugation for a minute. For IC ₅₀ determination, DE after cell lysis
To abolish VD (SEQ ID NO: 9) -caspase activity, 1 μM DEVD (SEQ ID NO: 9) -CHO (Biomol) was included in TET buffer.

Example 8 FRET Measurements Lysate corresponding to 1 mg total protein (transient transfection) or complete lysate (stable cell line) was diluted to 1 ml with 50 mM Tris, pH 7.5,
Fluorescence was measured using a Perkin Elmer LS 50B fluorometer. The level of FRET occurring between the two GFP chromophores was measured by the ratio of the intensity of the 527 nm emission peak (TOPAZ) to the intensity of the 476 nm emission peak (W1B) after excitation at 434 nm (W1B). No fluorescence was detected in Hela cells transfected with the pIRES1neo vector without insert.

Example 9 In Vitro Degradation of Tandem GFP Substrates The sequences encoding WT-1x and WT-2x were prepared using the existing EcoRI / BamH.
Using the I site, pIRES1neo to pcDNA3.1 (-) (Invi
trogen, Palo Alto, CA). Conjugate T
[ ³⁵ S] using the 7 wheat malt in vitro transcription / translation system (Promega)
Methionine labeled WT-1x and WT-2x proteins were produced. The pcDNA3.1 (−) construct (2 μg) was added to [ ³⁵ S] methionine (10 μCi / μl) (Amersham) in a total of 100 μl of wheat malt lysate.
8 μl and RNAsin (40 U / μl) (Boehringer Mannh
Incubated with 2 μl of eim) at 30 ° C. for 45 minutes. Degradation of radiolabeled WT-1x protein or WT-2x protein requires a final volume of 10 μl.
Degradation buffer (50 mM HEPES / KOH (pH 7.0), 2 mM EDT)
A, 0.1% CHAPS, 10% (w / v) glycerol, 10 mM DTT)
It was carried out by incubating with purified recombinant caspase-3 in 1 h at 3 ° C. The reaction was stopped by adding SDS sample buffer followed by heating at 95 ° C for 5 minutes. The degradation products were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The autoradiogram was quantified with a laser scanning densitometer.

Example 10 Western Blot Crude cell lysates (15 mg) were treated as previously described (Rasper et al., 1).
998, Cell Death and Differentiation 5
: 271-288), 10-20% Tris-glycine SDS gel separation and transfer to a nitrocellulose membrane. Immunoblot analysis for rabbit polyclonal anti-GF
Performed using a 1: 500 dilution of P antibody (Clontech, Palo Alto, CA). Donkey anti-rabbit peroxidase (Amersham) 1:
A 3000 dilution was used as the secondary antibody. The protein band was visualized by ECL detection (Amersham) and autoradiography.

Example 11 Generation of stable cell lines and 96-well plate assay To generate stable cell lines capable of expressing high levels of tandem GFP substrate, a tetracycline inducible expression system (Clontech, Palo Al.
to, CA) was used. PTRE-WT-1x and pTK in 60 mm dish
Hela T by Hyg (Clontech, Palo Alto, CA)
After co-transfection of et-On cells (Clontech, Palo Alto, CA), the cells were grown for an additional 48 hours and then passaged in 100 mm dishes in the presence of 150 μg / ml hygromycin. Pooled hygromycin resistant colonies for 48 hours to induce expression of the WT-1x fusion protein.
Treated with 2 μg / ml doxycycline. Fluorescent clones were individually isolated into 96-well plates using fluorescence activated cell sorting (FACS). One cell clone HETON-WT-1x-57 was selected for further analysis and 2 μg /
Costar black-wall 96-well clear bottom plates were seeded at a concentration of 4,000 cells / well in the presence of ml doxycycline. After 2 days of incubation, the medium was replaced with fresh medium containing doxycycline (2 μl / well). Staurosporine and various concentrations of zVAD-fmk were co-administered to cells (1.5% final DMSO) and incubated for 4 hours. Then, the medium is
Replace with a suitable filter set (420 / 50ex, 490 / 40em)
, And 530 / 25em) 96-well fluorometer (Perceptive)
Fluorescence from whole cells was measured using a Biosystems Cytofluor).

The present invention is not limited in scope by the particular embodiments described herein. Indeed, various embodiments of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are fully incorporated by reference.

[Brief description of drawings]

FIG. 1A is a diagram showing fluorescence resonance energy transfer between mutant GFP variants in a recombinant group II caspase substrate, and when donor GFP was excited at 434 nm, the released energy (476 nm) was transferred to acceptor GFP. And shows re-emission at 527 nm.

FIG. 1B is a diagram showing fluorescence resonance energy transfer between mutant GFP variants in a recombinant Group II caspase substrate, showing the respective fusion proteins (WT, WT-1x, WT) constructed under the schematic representation of tandem GFP protein. -2x, and WT-2xL) linker primary amino acid sequences are abbreviated.

FIG. 2A shows that FRET efficiency is a function of linker length, showing lysate emission scans (450-600 nm) following excitation at 434 nm.

FIG. 2B shows that FRET efficiency is a function of linker length, showing the measurement of FRET by ratiometric analysis.

FIG. 3 shows that tandem GFP substrates containing multiple DEVD (SEQ ID NO: 9) motifs are cleaved by caspase-3 more efficiently than substrates containing a single DEVD (SEQ ID NO: 9) motif. It is a figure.

FIG. 4A shows that apoptosis results in the degradation of DEVD (SEQ ID NO: 9) containing tandem GFP substrates, prepared from Hela cells transfected with the WT construct, the WT-1x construct, or the WT-2x construct. Figure 8 shows a Western blot analysis of tandem GFP substrate in lysates run.

FIG. 4B shows that apoptosis leads to the degradation of DEVD (SEQ ID NO: 9) containing tandem GFP substrates, in the presence (+) or absence (+) or non-specific caspase inhibitor (zVAD-fmk). -) From cells treated with staurosporine
4 shows a RET measurement.

[Figure 5]   FIG. 6 shows direct fluorescence quantification of caspase inhibition in whole cells.

FIG. 6A shows a high throughput screen for caspase inhibitors in multi-well microtiter plates containing a single compound.

FIG. 6B: High throughput screening of caspase inhibitors in multi-well microtiter plates containing drug mixture (10 compounds per well).

FIG. 7A shows the nucleotide sequence of wild-type green fluorescent protein (SEQ ID NO: 5).

FIG. 7B   It is a figure which shows the amino acid sequence of a wild-type green fluorescent protein (SEQ ID NO: 6).

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AU, CA, JP, US (72) Inventor Towa, Paul             Kebetsk Ashiyu, 9 reeds, Canada             You 3 El 1, Kirkland, Tran             Su-Canada Highway 16711 (72) Inventor Casadei, Robin             Kebetsk Ashiyu, 9 reeds, Canada             You 3 El 1, Kirkland, Tran             Su-Canada Highway 16711 (72) Inventor Nicholson, Donald             Kebetsk Ashiyu, 9 reeds, Canada             You 3 El 1, Kirkland, Tran             Su-Canada Highway 16711 F-term (reference) 4B024 AA01 AA11 BA80 CA02 CA05                       HA01 HA11 HA17                 4B063 QA01 QA19 QQ08 QQ36 QQ79                       QR16 QR48 QR67 QR77 QX02                 4H045 AA10 AA20 BA10 BA13 BA41                       CA50 EA20 EA50 FA74

Claims (13)

[Claims] 1. An ultra-bright green fluorescent protein (GFP) linked by a peptide containing at least one caspase degradation site.
A fusion protein comprising a donor GFP and an acceptor GFP that are 2. The fusion protein of claim 1, wherein the peptide containing at least one caspase cleavage site contains less than 10 amino acids and has an acceptor / donor fluorescence resonance energy transfer ratio of at least about 1.5. . 3. A DNA encoding the fusion protein of claim 1. 4. (a) Preparing a cell containing a fusion protein containing a donor green fluorescent protein (GFP) and an acceptor GFP linked by a peptide containing at least one caspase degradation site; (b) expression of caspase. The amount of fluorescence resonance energy transfer (FRET) in the cell in the absence of a substance suspected to be an activator of caspase; (c) a substance suspected to be an activator of caspase expression (D) measuring the amount of FRET in the cell in the presence of a substance suspected to be an activator of caspase expression, comprising: Therefore, the amount of FRET measured in step (b) is
A method wherein the substance is a caspase activator if it is greater than the amount of FRET measured in). 5. (a) Preparing a cell containing a fusion protein containing a donor green fluorescent protein (GFP) and an acceptor GFP linked by a peptide containing at least one caspase degradation site; (b) activating caspase. (C) measuring the amount of fluorescence resonance energy transfer (FRET) in the treated cells in the absence of a substance suspected of being a caspase inhibitor; (d) Exposing treated cells to a substance suspected of being a caspase inhibitor; (e) measuring the amount of FRET in treated cells exposed to a substance suspected of being a caspase inhibitor. A method for identifying a caspase inhibitor comprising: wherein the amount of FRET measured in step (e) is
The method wherein the substance is a caspase inhibitor if it is greater than the amount of FRET measured in. 6. (a) Preparing a cell containing a fusion protein containing a donor green fluorescent protein (GFP) and an acceptor GFP linked by a peptide containing at least one caspase degradation site; (b) activating caspase. (C) measuring the amount of fluorescence resonance energy transfer (FRET) in the treated cells in the absence of a substance suspected of being a caspase inhibitor; (d) Exposing the treated cells to a substance suspected to be a caspase inhibitor, wherein the treated cells are exposed to at least about 5,000 different substances for 24 hours, (e ) Measuring the amount of FRET in treated cells exposed to a substance suspected to be a caspase inhibitor comprising: A method of high throughput screening for identification, wherein the substance is a caspase inhibitor when the amount of FRET measured in step (e) is greater than the amount of FRET measured in step (c). Method. 7. The method of claim 6, wherein the cell is a eukaryotic cell. 8. The method of claim 6, wherein the cells are mammalian cells. 9. The cells are L cells LM (TK ⁻ ) (ATCC CCL 1.
3), L cells LM (ATCC CCL 1.2), HEK293 (ATCC
CRL 1573), Raji (ATCC CCL 86), CV-1 (ATC
C CCL 70), COS-1 (ATCC CRL 1650), COS-7
(ATCC CRL 1651), CHO-K1 (ATCC CCL 61),
3T3 (ATCC CCL 92), NIH / 3T3 (ATCC CRL 16
58), Hela (ATCC CCL 2), C127I (ATCC CRL)
1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC
CCL 171), PC12 cells, C. elegans cells, zebrafish cells, Xenopus oocytes,
Xenopus melanophore and Saccharomyces cerevisiae (Saccha
7. The method of claim 6, selected from the group consisting of Romyces cerevisiae) cells. 10. The donor GFP is a super bright GFP, FRET is measured by ratiometric analysis, the cells are adherent mammalian cells, and the peptide containing the caspase degradation site contains less than 10 amino acids. The method of claim 6, wherein 11. A caspase degradation site is DXXD (SEQ ID NO: 7), where X is any naturally occurring amino acid; DEXD (SEQ ID NO: 8), wherein X is any naturally occurring amino acid. DEVD (SEQ ID NO: 9); DEHD (SEQ ID NO: 10); DETD (SEQ ID NO: 11); DGPD (SEQ ID NO: 12); DEPD (SEQ ID NO: 13); DELD (SEQ ID NO: 14); DAVD (SEQ ID NO: 15) DMQD (SEQ ID NO: 16); DSID (SEQ ID NO: 17); DVPD (SEQ ID NO: 18); DQTD (SEQ ID NO: 19); DSLD (SEQ ID NO: 20); WEHD (SEQ ID NO: 21); ZEXD (SEQ ID NO: 22) here Where X is any naturally occurring amino acid and Z is either I, L or V; LEHD (SEQ ID NO: 23); VEH D (SEQ ID NO: 24); LETD (SEQ ID NO: 25); YVAD (SEQ ID NO: 26); LVAD (SEQ ID NO: 27); ELPD (SEQ ID NO: 28); SRVD (SEQ ID NO: 29); VEID (SEQ ID NO: 30); and 11. The method of claim 10, selected from the group consisting of IETD (SEQ ID NO: 31): 12. The method according to claim 10, wherein the caspase degradation site is a caspase-2 degradation site, a caspase-3 degradation site, or a caspase-7 degradation site. 13. The method of claim 10, which is carried out in a microtiter-like plate having 96, 384, or 1,536 wells per plate, wherein the superluminescent GFP is W1B; TOPAZ; 10C. ; 10C Q69K; W7
W1C; wild type GFP with changes S65G, S72A, and T203F; and wild type GFP with changes S65G, S72A, and T203H (SEQ ID NO: 6): well volume less than 250 μl, 25 μl Less than, or less than 10 μl.