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WO2000073437A1 - Analyses permettant de detecter l'activite de caspases a l'aide de proteines fluorescentes vertes - Google Patents

Analyses permettant de detecter l'activite de caspases a l'aide de proteines fluorescentes vertes Download PDF

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Publication number
WO2000073437A1
WO2000073437A1 PCT/CA2000/000620 CA0000620W WO0073437A1 WO 2000073437 A1 WO2000073437 A1 WO 2000073437A1 CA 0000620 W CA0000620 W CA 0000620W WO 0073437 A1 WO0073437 A1 WO 0073437A1
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Prior art keywords
caspase
cells
seq
gfp
fret
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PCT/CA2000/000620
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English (en)
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Steven Xanthoudakis
Paul Tawa
Robin Cassady
Donald Nicholson
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Merck Frosst Canada & Co.
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Priority to AU49061/00A priority Critical patent/AU4906100A/en
Priority to EP00930934A priority patent/EP1185644A1/fr
Priority to JP2001500750A priority patent/JP2003501024A/ja
Priority to CA002374369A priority patent/CA2374369A1/fr
Publication of WO2000073437A1 publication Critical patent/WO2000073437A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43595—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)

Definitions

  • the present invention is directed to methods for determining the activity of caspases.
  • the methods utilize fluorescent resonance energy transfer between two green fluorescent proteins linked as a fusion protein having a caspase cleavage site between them.
  • the methods can be used to identify substances that are activators or inhibitors of caspase activity either in living cells or in vitro.
  • Apoptosis is characterized by distinct series of morphological and biochemical changes that inevitably result in the death of a cell (Kerr et al., 1972, Br. J. Cancer 26:239-25 " : Vaux et al., 1994, Cell 76:777-779; whilr, 1995, Science 267:1445-1449). Apoptosis occurs during developmental morphogenesis, in the removal of expended, unnecessary or irreparably damaged cells, and in response to pathogenic infections (Kerr et al., 1972, Br. J. Cancer 26:239-257; McConkey et al., 1996, Mol. Aspects Med. 17:1-110; whilr, 1995, Science 267:1445-1449; Thompson, 1995.
  • Apoptosis proceeds through a highly regulated series of biochemical events and many of the components of the cell death pathway have been identified.
  • biochemical mediators of apoptosis are a group of proteolytic enzymes known as the caspases, which serve to disrupt normal cellular homeostasis by cleaving specif::: target proteins with either structural, regulatory or housekeeping functions (Thornberry & Lazebnik, 1998, Science 281: 1312-1316; Thornberry, 1997, Br. Med. Bull. 53:478-490; Nicholson & Thornberry, 1997, Trends Biochem. Sci. 22:299-306).
  • the caspases comprise a large family of proteins that can be distinguished according to their substrate specificities (Thornberry, 1997, J Biol Chem 272:17907-17911). Twelve caspases have so far been identified in human cells. All caspases are synthesized as dormant proenzymes and can be rapidly activated through proteolytic maturation (Thornberry & Lazebnik, 1998, Science 281:1312-1316; Thornberry, 1997, Br. Med. Bull. 53:478-490). Members of the caspase family can be divided into three functional subgroups based on their substrate specificities which have been defined by a positional-scanning combinatorial substrate approach (Rano et al., 1997, Chem. Biol.
  • caspases As therapeutic targets for various disease processes (e.g., acute neurodegeneration, cardiac ischemia, hepatic failure) (Nicholson, 1996. NaT- Biotechnol. 14:297-301).
  • methods for monitoring caspase activity are primarily limited to analyzing downstream biochemical events (e.g., DNA fragmentation, membrane remodeling) that are often several steps removed from the proteolysis caused by active caspases (Darzynkiewicz & Traganos, 1998, Adv. Biochem. Eng. Biotechnol. 62:33-73).
  • GFP-based reporter strategies have been used in numerous cellular systems to monitor transcriptional activation, cellular localization and protein trafficking (Misteli et al., 1997. Nat. Biotechnol. 15:961-964; Tsien, 1998, Annu. Rev. Biochem. 67:509-544). Their utility as in vitro substrates for protease assays have also been demonstrated fPollok & Heim, 1999, Trends Cell Biol. 9:57-60; Mitra et al., 1996, Gene 173: 13-17: Heim et al., 1996, Curr. Biol. 6:178-182). Xu et al., 1998, Nucl. Acids Res.
  • 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 was used to monitor the activation of caspase activity by the use of fluorescence activated cell sorting (FACS) as well as by detection of fragments of the fusion protein by Western blot. Xu did not disclose methods of high-throughput screening for identifying inhibitors of caspases. Heim & Tsien.
  • the present invention is directed to methods of monitoring caspase activity by utilizing fluorescent resonance energy transfer between two green fluorescent proteins linked as a fusion protein having a caspase cleavage site between them.
  • the methods are fast, sensitive, and generally act at the level of caspase activity, i.e., proteolysis of caspase substrates, rather than at the level of downstream events.
  • the methods can be practiced in vitro, e.g., using cell-free extracts or purified components, or the methods can be practiced in vivo, i.e., in living cells.
  • the methods can be adapted to provide assays suitable for the identification of activators as well as inhibitors of caspase activity.
  • Figure 1 A-B shows fluorescence resonance energy transfer between mutant GFP variants in a recombinant group II caspase substrate.
  • Figure 1A Upon excitation of the donor GFP (W1B) at 434 nm, its emitted energy (476 nm) is transferred to the acceptor GFP (TOPAZ) and is re-emitted at 527 nm. This energy transfer is dependent on the close physical proximity of the two GFP proteins.
  • FIG. 1B Outlined below the schematic of the tandem GFP protein is the primary amino acid sequence of the linker for each of the fusion proteins constructed (WT, WT-lx, WT-2x, and WT-2xL). Note that the WT fusion contains a short six amino acid linker lacking a caspase tetrapeptide cleavage motif.
  • the WT-lx and WT-2x proteins contain a single and two tandem group II caspase cleavage motifs (DEVD (SEQ.ID.NO. :9)) within the linker, respectively.
  • WT-2xL contains two tandem DEVD (SEQ.ID.NO. :9) cleavage motifs flanked by six amino acid SG repeats designed to confer flexibility and accessibility 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-lx linker is SEQ.ID.NO. :2;
  • the amino acid sequence shown for the WT-2x linker is SEQ.ID.NO. :3;
  • the amino acid sequence shown for the WT-2xL linker is SEQ.ID.NO. :4.
  • Figure 2A-B shows that FRET efficiency is a function of the linker length. Lysates from Hela cells transfected transiently with constructs encoding the WT, WT-lx, or WT-2xL fusion proteins were assayed for fluorescence.
  • Figure 2A shows the emission scan (450-600 nm) of the lysates following excitation at 434 nm.
  • Figure 3 shows that tandem GFP substrates containing multiple DEVD (SEQ.ID.NO. :9) motifs are cleaved more efficiently by caspase-3 than substrates containing a single DEVD (SEQ.ID.NO. :9) motif.
  • SEQ.ID.NO. :9 Single DEVD
  • FIG. 3 shows that tandem GFP substrates containing multiple DEVD (SEQ.ID.NO. :9) motifs are cleaved more efficiently by caspase-3 than substrates containing a single DEVD (SEQ.ID.NO. :9) motif.
  • In vitro translated and [35S]methionine- ⁇ abe! ⁇ d WT-lx and WT-2x proteins were incubated with varying concentrations of punfied recombinant caspase-3 for 1 hr at 37°C.
  • Cleaved proteins were resolved by SDS polyacrylamide gel electrophoresis and quantitated by autoradiography and scanning densitometry of the gel
  • Figure 4A-B shows that apoptosis results in cleavage of DEVD (SEQ.ID.NO. :9)-conta ⁇ nmg tandem GFP substrates.
  • Figure 4A shows Western blot analysis of the tandem GFP substrates in lysates prepared from Hela cells transfected with the WT, WT-1 ⁇ . or WT-2x constructs. Apoptosis was induced by treatment of the cells for 4 hrs with 1 ⁇ M staurosponne.
  • Figure 4B shows FRET measurements from cells treated with staurosponne in the presence (+) or absence (-) of a pan caspase inhibitor (zVAD-fmk) Note that FRET is diminished in apoptotic extracts containing caspase-3 sensitive tandem GFP substrates only.
  • Figure 5 shows the direct fluorometnc measurement of caspase inhibition in intact cells.
  • Hela cells clone HETON-WT-lx-57
  • WT-lx tandem GFP substrate were treated simultaneously with staurosponne to induce apoptosis and ⁇ ary g concentrations of zVAD-fmk for 4 hrs.
  • Cells were assayed for FRET (530 em./490 em), after exchanging the media for PBS.
  • the 0.01 ⁇ M and 10,000 ⁇ M titration points represent values for the maximum (DMSO vehicle alone) and minimum (staurosponne alone) FRET ratios obtained, respectively.
  • Figure 6A-B shows high-throughput screening of caspase inhibitors in multiwell microtiter plates containing either single compounds ( Figure 6A) or drug mixtures (10 compounds/well) ( Figure 6B).
  • Duplicate 96-well plates were co-treated with staurosponne and drug(s) for four hours.
  • Three concentrations of zVAD-fmk (2.5 ⁇ M, 25 ⁇ M. and 250 ⁇ M) were randomly added to two wells in each plate. The experiment was performed blindly, such that the well location of zVAD-fmk was not revealed until the data were processed.
  • the black and gray bars indicate the position of the wells containing 250 ⁇ M and 25 ⁇ M zVAD-fmk, respectively.
  • FIG. 7A-B shows the nucleotide ( Figure 7A, SEQ.ID.NO.:5) and the amino acid ( Figure 7B. SEQ.ID.NO. :6) sequences of wild-type green fluorescent protein.
  • a “green fluorescent protein (GFP)” is a fluorescent protein in which any 150 contiguous amino acids have an amino acid sequence identity of at least 85% to a contiguous stretch of the amino acid sequence of wild-type GFP (SEQ.ID.NO. :6).
  • An '"ultra-bright green fluorescent protein (GFP)” is a GFP that has been engineered to contain amino acid changes from wild-type GFP that result in enhanced emission properties compared to wild-type GFP.
  • Ultra-bright GFPs do not have the change Y66H.
  • Ultra-bright GFPs have a fluorescence quantum yield of at least 0.39.
  • ultra-bright GFPs are W1B; TOPAZ; 10C; 10C Q69K; W7; WIC; wild-type GFP (SEQ.ID.NO.:6) having the changes S65G, S72A, and T203F; and wild-type GFP (SEQ.ID.NO.:6) having the changes S65G, S72A, and T203H.
  • High-throughput screening refers to methods of identifying activators or inhibitors of a biological activity, e.g., the activity of a caspase, in which a large number of substances suspected of having a desired property, e.g., being an activator or an inhibitor of a caspase, are tested to determine if in fact such substances have such a property .
  • High-throughput screening involves the testing of at least 5,000, preferably at least 10,000, and even more preferably at least 100,000 substances per 24 hour period to determine if any of the substances have the desired property.
  • the methods of high-throughput screening are carried out in volumes of less than 250 ⁇ l, preferably less than 25 ⁇ l, and even more preferably less than 10 ⁇ l in microtiter-like plates having at least 96 wells, preferably at least 384 wells, and even more preferably at least 1,536 wells per plate.
  • “Ratiometric analysis” refers to measurement of fluorescence resonance energy transfer (FRET) by determining the ratio of the fluorescence emitted by an acceptor green fluorescent protein (e.g., TOPAZ, 527 nm) divided by the fluorescence emitted by a donor green fluorescent protein (e.g., W1B, 476 nm) after excitation of the donor green fluorescent protein.
  • FRET fluorescence resonance energy transfer
  • Fusion protein refers to a polypeptide comprising a sequence of amino acids in which two green fluorescent proteins (GFPs) are joined by a linker that consists of a short stretch of ammo acids where the linker compnses at least one caspase cleavage site
  • GFP green fluorescent proteins
  • One GFP is a donor GFP and the other GFP is an acceptor GFP.
  • the two GFPs are different GFPs such that fluorescence resonance energy transfer (FRET) can occur from the donor GFP to the acceptor GFP when the linker is intact but, upon cleavage of the linker, FRET is abolished or greatly diminished
  • caspase inhibitor refers to a substance that decreases the proteolytic activity of a caspase protein A caspase inhibitor has no necessary effect on the amount of caspase protein produced by a cell Nor does a caspase inhibitor necessanly act at the level of proteolysis by a caspase A caspase inhibitor may decrease the proteoKtic activity of a caspase protein by acting at the level of upstream events that result in the activation of the caspase For example, a caspase inhibitor may act to prevent the release of cytochrome c from mitochondna, thus preventing the activation of a caspase
  • the methods of the present invention provide means for identifying caspase inhibitors Once identified, such inhibitors can be further charactenzed with respect to the level at which they act.
  • caspase activator refers to a substance that increases the proteolytic activity of a caspase prote .
  • a caspase activator has no necessary effect on the amount of caspase prote produced by a cell.
  • a caspase activator necessanly act at tne level of proteolysis by a caspase.
  • a caspase activator may increase the proteohtic activity of a caspase protein by acting at the level of upstream events that result m tne activation of the caspase.
  • the level at which a caspase activator acts can be determined by suitable in vitro assays.
  • FRET fluorescence resonance energy transfer
  • FRET is a process in which energy is transferred in a non-radiative manner from an excited donor fluorescent reagent to an acceptor fluorescent reagent by means of intermolecular long-range dipole-dipole coupling.
  • FRET typically occurs over distances of about 10A to lOOA and requires that the emission spectrum of the donor reagent and the absorbance spectrum of the acceptor reagent overlap adequately and that tne quantum yield of the donor and the absorption coefficient of the acceptor be sufficiently high.
  • the transition dipoles of the donor and acceptor fluorescent reagents must be properly oriented relative to one another.
  • the present invention is directed to fusion proteins comprising two green fluorescent proteins (GFPs) linked by a peptide comprising at least one caspase cleavage site and uses of the fusion proteins in methods of identifying substances that are activators or inhibitors of caspase activity either in cells or in vitro.
  • the fusion proteins comprise a donor GFP and an acceptor GFP. FRET occurs between the donor GFP and the acceptor GFP when the donor GFP is excited and when the linker between the donor GFP and the acceptor GFP is intact. Caspase cleavage of the linker separates the donor and acceptor GFPs, thus abolishing FRET between them.
  • the fusion proteins preferably are derived from GFPs having altered, enhanced spectral properties as compared with wild-type GFPs.
  • GFPs are known in the art (see. e.g., U.S. Patent No. 5,625,048; International Patent Publication WO 97/28261; International Patent Publication WO 96/23810).
  • GFPs W1B and TOPAZ available commercially from Aurora Biosciences Corp., San Diego. CA.
  • W1B contains the following changes from the wild-type GFP sequence: F64L, S65T. Y66W, N146I, M153T, and V163A (see Table 1, page 519, of Tsien, 1998, Ann. . Biochem. 67:509-544).
  • TOPAZ contains the following changes from the wild-type GFP sequence: S65G, V68L, S72A, and T203Y (see Table 1, page 519. of Tsien, 1998, Ann. Rev. Biochem. 67:509-544). Wild-type nucleotide and ammo acid sequences of GFP are shown in Figure 1 and SEQ.ID.NO.: 1 of International Pa ⁇ nt Publication WO 97/28261 ; in Figure 1 of Tsien, 1998, Ann. Rev. Biochem. 67:509-544; in Prasher et al., 1992, Gene 111:229-233; and in Figure 7A-B of this application.
  • the ch ⁇ . ⁇ of GFPs to use in the fusion protein should be guided by certain general considerations.
  • the excitation spectra of the donor and acceptor GFP should overlap as little as possible. This will allow the donor to be excited without directly exciting the acceptor.
  • 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 the donor and acceptor should overlap as little as possible.
  • the quantum yield of the donor and the extinction coefficient of the acceptor should be as high as possible.
  • Either the donor or the acceptor GFP may be at the amino or the carboxy terminal portion of the fusion protein.
  • additional peptide sequences may be present at the amino or carboxy terminal ends of the fusion proteins.
  • suitable fusion proteins of the present invention may be represented by the following schematics:
  • the intact fusion proteins preferably are capable of FRET to the extent that ratiometric anahsis will show a FRET ratio of at least 1.5, preferably at least 2.0. 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 and preferabh at least 2.0. Such ratios are especially suitable when using the methods of the present invention as a high-throughput screening tool since such ratios permit sensitive detection of caspase activation and allow for the exploitation of the advantages of ratiometric analysis, as described herein.
  • the linker is a peptide, i.e., a short stretch of amino acids, of length between about 4 and 50 amino acids. Preferred are linkers of length 10 amino acids or fewer, including the caspase cleavage site.
  • the linker comprises a single caspase cleavage site or multiple caspase cleavage sites that may be the same or different. Generally, the caspase cleavage site is chosen so that it is recognized by a single caspase. However, for some purposes, it may be preferable to use cleavage sites that are recognized by more than one caspase. It may even be advantageous to employ a linker that comprises more than a single caspase cleavage site where the cleavage sites are recognized by different caspases. Such linkers are useful when it is desired to assay for activators or inhibitors of more than a single caspase.
  • Methods of identifying activators and inhibitors of caspases in living cells are provided b> the present invention. These methods generally involve the use of cells that have been engineered to express a fusion protein compnsing two green fluorescent proteins (GFPs) linked by a peptide compnsing at least one caspase cleavage site.
  • GFPs green fluorescent proteins
  • Such cells are produced by transfectmg cells with expression vectors compnsing DNA encoding the fusion proteins
  • the amount of fluorescence resonance energy transfer (FRET) in the engineered cells from the donor GFP in the fusion protem to tne acceptor GFP is determined Preferably, this determination is done by means of ratiometnc analysis.
  • FRET fluorescence resonance energy transfer
  • the engineered cells are exposed to a substance that is suspected of being an activator of a caspase If the substance is actually an activator, this results m an increase in caspase activity within the cells, leading to cleavage of the linker of the fusion protein.
  • the tw o GFPs making up the fusion protein are then free to dnft apart, decreasing tne amount of FRET between them. This decrease in FRET is measured, preferaoh ratiometnc analysis.
  • Methods of identifying inhibitors of caspases are similar, except that while the cells that are treated to induce caspase activity and/or apoptosis.
  • the present invention includes a method of identifying a caspase activator compnsing:
  • the present invention also includes a method of identifying a caspase inhibitor comprising:
  • the cells are eukaryotic cells.
  • the cells are mammalian cells or are yeast cells.
  • the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2).
  • HEK293 ATCC CRL 1573
  • Raji ATCC CCL 86
  • CV-1 ATCC CCL 70.
  • COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), Hela (ATCC CCL 2), C12T (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171 .. PC12 cells.
  • the cells are cells within a whole, living organism such as , e.g., the nematode C. elegans or the zebrafish.
  • the fusion proteins are introduced into the organism by methods well known in the art.
  • An advantage of using C. elegans and zebrafish is that these organisms are translucent, making observation of FRET in a living animal possible. In this way, the activity of caspase activators and inhibitors can be monitored in real time in a whole organism.
  • cells can be prepared from these organisms that comprise the fusion protein and such cells can be used to practice the methods of the invention.
  • the present invention makes use of trangenic animals, e.g..
  • transgenic mice in which at least some cells express a fusion protein comprising a donor and an acceptor green fluorescent protein (GFP) linked by a peptide comprising at least one caspase cleavage site.
  • GFP green fluorescent protein
  • Such transgenic animals are useful in understanding the role of caspases in conditions such as, e.g., cardiovascular diseases, neurodegenerative diseases, traumatic or septic shock, and stroke.
  • a transgenic mouse expressing the fusion protein in its neural tissue is a useful model for assessing the effects of caspase inhibitors on a chronic or acute insult, e.g., neurodegenerative diseases, brain trauma, or stroke. In this model as applied to stroke, one would induce stroke in the transgenic mouse in the presence and in the absence of a caspase inhibitor.
  • the brain tissue of the mouse would then be sectioned and imaged by microscopy to determine if the caspase inhibitor is reaching particular areas of the brain and is inhibiting a caspase in those areas. Analysis of the effects of the inhibitor combined with analysis of stroke damage can determine whether there is a correlation between caspase inhibition and protection from stroke damage.
  • the present invention can therefore be used to evaluate the ability of caspase inhibitors to protect brain tissue from damage due to stroke.
  • the cells are adherent cells rather than suspension cells.
  • the methods are practiced in the wells of a microtiter-like plate and the cells are not harvested or detached from the wells, i.e., the cells remain attached to the bottoms of the wells throughout the practice of the method.
  • an expression vector encoding the fusion protein is transfected into the cells.
  • Such an expression vector can be mace bv well-known recombinant DNA methods such as those described in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press; or PCR Primer, A Laboratory Manual, edited by C.W. Dieffenbach and G.S. Dveksler, 1995, Cold Spring Harbor Laboratory Press.
  • One method would involve starting with a standard expression vector.
  • DNA sequences encoding the donor and acceptor GFPs are cloned into the polylinker site of the standard expression vector, preferably after PCR of the GFPs so that their sequences are flanked by restriction enzyme cleavage sites. After the GFPs have been cloned into the expression vector, there will be a restriction site or sites between the GFP sequences into which the linker can be cloned.
  • the linker sequence is chosen so that the donor GFP, acceptor GFP, and linker are all in the same reading frame in the final expression construct.
  • a fusion protein comprising two GFPs separated by a 20 amino acid linker having a protease site (in this case a Factor Xa site) are described in Mitra et al., 1996, Gene 173: 13-17.
  • International Patent Publication WO 97/28261 at Example 1, beginning at page 40, discloses methods for constructing GFP fusion proteins containing linkers having a variety of protease cleavage sites. The general procedures disclosed in these references can be used as guidelines for constructing the caspase-specific fusion proteins of the present invention.
  • Transfection can be accomplished by any of the methods known in the art, e.g., calcium phosphate or calcium chloride mediated transfection, electroporation, infection with a retroviral vector.
  • the cells can be transiently or stably transfected.
  • the present invention includes DNA encoding fusion proteins comprising two green fluorescent proteins (GFPs) linked by a peptide comprising at least one caspase cleavage site.
  • the present invention also includes expression vectors comprising DNA encoding fusion proteins comprising two green fluorescent proteins (GFPs) linked by a peptide comprising at least one caspase cleavage site as well as host cells comprising these expression vectors.
  • a variety of standard expression vectors can be used to produce the expression vector encoding the fusion protein.
  • Commercially available expression vectors which are suitable include, but are not limited to, pMClneo (Stratagene, La Jolla, CA), pSG5 -Stratagene, La Jolla, CA), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3J, pCR3J (Tnvitrogen, Palo Alto, CA), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), and pIRESlneo (Clontech.
  • Another way of improving GFP expression in mammalian cells is to provide an optimal ribosome binding site by the use of an additional codon immediately after the starting methionine (Crameri et al., 1996, Nature Biotechnology 14:315-319).
  • an expression vector in which the GFP fusion protein can be cloned under the control of an inducible promoter such as, e.g.. promoters induced by dexamethasone, metallothionein. or tetracycline and related analogs such as doxycycline.
  • an inducible promoter such as, e.g.. promoters induced by dexamethasone, metallothionein. or tetracycline and related analogs such as doxycycline.
  • a suitable vector containing an inducible promoter is the pTRE vector from Clontech, Palo Alto, CA; this vector contains a tetracycline inducible promoter.
  • Other such vectors are well-known in the art and many are available commercially.
  • the donor and acceptor GFPs are selected from the group consisting of:
  • W1B wild-type GFP (SEQ.ID.NO.:6) having the changes F64L, S65T, Y66W, N146I, M153T. and V163A;
  • EGFP wild-type GFP (SEQ.ID.NO.:6) having the changes F64L and S65T;
  • wild-type GFP (SEQ.ID.NO.:6) having the changes T203I, S72A, and Y145F: IOC Q69K
  • wild-type GFP (SEQ.ID.NO. :6) having the changes S65G
  • wild-type GFP (SEQ.ID.NO. :6) having the changes S65A, Y66W, S72A, N146L M153T, and VI 63 A;
  • wild-type GFP (SEQ.ID.NO.:6) having the change Y66H and Y145F;
  • EBFP wild-type GFP (SEQ.ID.NO. :6) having the change F64L, Y66H, and Y145F.
  • the step of treating the cells in order to activate a caspase is performed by: viral infection; growth factor withdrawal; anticancer agents; exposure to ultraviolet radiation: treatment with staurosporine, actinomycin D, etoposide, glucocorticoids, camptothecin. thapsigargin, cyclohexamide, rapamycin, ceramide, glutamate, N-methyl-D-aspartate, kainic acid, lectins, tributyltin, vincristine, vinblastine, cisplatin.
  • TNFR tumor necrosis factor receptor
  • the step of measuring the amount of FRET is performed by ratiometric analysis. In certain embodiments, the step of measuring the amount of FRET is performed by measuring an increase or decrease in the emission of the donor GFP. In certain embodiments, the step of measuring the amount of FRET is performed by measuring an increase or decrease in the emission of the acceptor GFP.
  • the conditions under which the step of exposing the cells to the substance is practiced are conditions that are typically used in the art for the study of protein-ligand interactions or exposure of cells to compounds: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media: a temperature of about 4°C to about 55°C. In certain embodiments, it may be advantageous to include a substance that quenches background fluorescence in the tissue culture media in which the cells are grown.
  • the above-described methods are practiced as methods of high-throughput screening.
  • the methods are repeated at least 5.000. preferably at least 50,000, even more preferably at least 100,000 times in 24 hour period.
  • at least 5,000, preferably at least 50.000, even more preferably at least 100,000 different substances are tested in a 24 hour period.
  • the methods are carried out in microtiter-like plates.
  • the microtiter-like plates have 96 wells, 384 wells, 1.536 wells, 3,064 wells, 3,456, wells, or 9,600 wells.
  • step (c) does not comprise transfecting the cells with a gene that induces apoptosis or caspase activation and the step of measuring FRET does not include FACS analysis or flow cytometry.
  • the caspase is a mammalian caspase.
  • the caspase is a human caspase.
  • the caspase is selected from the group consisting of: caspase- 1 (also known as interleukin-l ⁇ converting enzyme (ICE)) (Thornberry et al.. 1992.
  • caspase- 1 also known as interleukin-l ⁇ converting enzyme (ICE)
  • caspase-2 also known as ICH-1 (Wang et al., 1994, Cell 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. 3:619-625); caspase-4 (also known as ICE re l-II, TX, ICH-2) (Munday et al., 1995,
  • caspase-7 also known as Mch3, ICE-LAP3, CMH-1 (Fernandes- Alnemri et al., 1995. Cancer Res. 55:6045-6052
  • caspase-8 also known as MACH, FLICE, Mch5 (Boldin et al., 1996,
  • the caspase cleavage site comprises an amino acid sequence selected from the group consisting of:
  • DXXD (SEQ.ID.NO. :7), where X is any naturally occurring amino acid
  • DEND SEQ.ID. ⁇ O.:9; DEHD (SEQ.ID.NO.: 10); DETD (SEQ.ID.NO.: 11);
  • DGPD (SEQ.ID.NO.: 12);
  • DMQD (SEQ.ID.NO.: 16);
  • DNPD (SEQ.ID.NO.: 18);
  • DQTD SEQ.ID.NO.: 19
  • DSLD SEQ.ID.NO.:20
  • ZEXD (SEQ.ID.NO. :22), where X is any naturally occurring amino acid and Z is either I. L. or V;
  • LEHD SEQ.ID.NO.:23
  • VEHD SEQ.ID.NO.:24
  • VEID (SEQ. ⁇ D.NO.:30).
  • EETD (SEQ.ID.NO.:31).
  • cleavage site preferences of a variety of caspases. This publication describes methods of identifying capsase cleavage sites by the use of a positional scanning combinatorial library.
  • suitable caspase cleavage sites for use in the present invention include any sequences that are determined to be caspase cleavage sites by such methods or that are known in the art to be caspase cleavage sites.
  • the caspase for which an activator or an inhibitor is identified is a caspase that is naturally expressed in the cell.
  • the caspase for which an activator or an inhibitor is identified is provided by transfecting into the cells an expression vector that directs the expression of the caspase. This can be done in order to provide for expression of the caspase in cells in which the caspase is not naturally expressed (e.g., in yeast or C. elegans.
  • the caspase that is transfected into the cells is a caspase that contains mutations or alterations in its amino acid sequence as compared to a wild-type caspase
  • the caspase is denved from a species other than the species from which the cells are denved.
  • tne absence of other components of the apoptotic pathway means that any effect caused by the substance on caspase activity (as measured by changes in FRET) will be due to a direct effect on the caspase itself
  • An example of such an in vitro assay is descnbed in Examples 2 and 9 and Figure 4 herein.
  • Anotner way to determine the level at which a caspase inhibitor acts is to allow a penod of time to pass between step (b) of the method (treating the cells in order to activate a caspase) and step (d) (exposing the cells to a substance suspected of being a caspase inhibitor). This penod of time will allow for active caspase to be generated.
  • This acuv e caspase will result in a large amount of cleavage of the fusion protein, and thus httie FRET.
  • the substance is an inhibitor
  • exposing the cells to the substance will increase the amount of FRET
  • the inhibitor acts at the level of caspase proteolysis. This increase should occur quickly, on the order of the amount of time it takes the inhibitor to enter the cells.
  • the inhibitor acts on upstream events, e.g., if it acts to turn off upstream caspase activation events, then the effect of the inhibitor on caspase proteolysis should occur more slowly. In such a case, the increase in FRET should occur more slowly as well.
  • step (b) The amount of time it takes for the treatment of step (b) to actually activate the caspase will vary depending upon a number of factors such as the type of cell used in the method, the nature of the treatment (e.g., the identity of a compound used to activate the caspase), and the concentration of the compound that is used to activate the caspase. Some compounds can activate a caspase and/or induce apoptosis very quickly (i.e.. on the order of minutes), while others take hours or even overnight incubation. In most cases, the treatment that activates the caspase should be carried out for about 2 to S hours before the cells are exposed to the substance suspected of being a caspase inhibitor, if it is desired to obtain knowledge of the level at which the substance acts.
  • the cells may be exposed to the substance concurrently with or prior to the application of the treatment that activates a caspase. This will depend on factors such as the metabolic stability of the substance, cell permeability, or even the kinetic characteristics of the substance/caspase interaction, e.g., the on rate, affinity, K[.
  • Measuring the amount of FRET is generally done by exciting the donor GFP by use of a lamp, laser, or other light source tuned to the donor GFP's excitation wavelength.
  • the excitation energy absorbed by the donor can be emitted by the donor at its emission wavelength or the excitation energy can be transferred, at least partially, to the acceptor GFP which can in turn emit the excitation energy at its own emission wavelength.
  • the acceptor GFP can in turn emit the excitation energy at its own emission wavelength.
  • ratiometric analysis is preferred because the magnitude of the ratio of donor acceptor emission is a measure of the cleavage of the fusion protein that is not influenced by such factors as the absolute amount of the fusion protein or its cleaved products, the optical thickness of the sample being assayed, the brightness of the excitation source, or the sensitivity of the detector.
  • ratiometnc analysis also is preferred because ratiometnc analysis affords a level of sensitivity that is suited for use in high-throughput screening methods
  • the sensitivity of the methods of the present invention is also increased by the use of ultra-bnght GFPs as donor GFPs Rather than employing ultra-bnght GFPs.
  • the pnor art employed a type of GFP known as a blue fluorescent protein (BFP) as a donor GFP (Xu et al., 1998, Nucl. Acids Res. 26:2034-2035 (Xu)).
  • BFPs have a mutation at position 66 that changes the wild-type tyrosine at that position to a histidine This Y66H change results in a protein having relatively weak fluorescence (i.e..
  • Hi h-tnroughput screening requires sample throughputs that exceed the present capacity of such methods as flow cytometry.
  • Flow cytometry requires such steps as harvesting cells, washing the cells, feeding the cells into a su ⁇ tab:e apparatus, and then analyzing the cells one at a time.
  • High- throughput screening is preferably done in multi-well microtiter-like plates rather than in flow cytometrv instrumentation. Multi-well plates afford the possibility of parallel analysis of thousands of assays every few minutes, whereas flow cytometry permits the analysis of at most hundreds of assays per minute (see Nolan et al., 1999, Drug Discovery Today 4:173-180. at page 179, right column).
  • a variety of automated analysis equipment that can be used with microtiter-like plates and that is suitable for carrying out high-throughput screening is known. See, e.g., U.S. Patent No. 5,670,113; U.S. Patent No. 5.139,744; and U.S. Patent No. 4,626,684.
  • the methods of the present invention provide several advantages compared to the prior art that make these methods especially suitable for high- throughput screening.
  • the use of ratiometric analysis with ultra-bright GFPs obviates the cumbersome and time consuming step of harvesting cells from the multi-well plates, as required in flow cytometry methods such as, e.g., FACS analysis.
  • the methods of the present invention are especially suitable for use in adherent cells such as Hela cells, 293 cells. L cells. CHO cells, PC12 cells, COS cells, or 3T3 cells.
  • the methods described herein by obviating the need for such cumbersome steps as harvesting adherent cells from the multi-well plates, provide means of screening vastly more compounds than can be done using less efficient flow cytometry methods, such as those described in Xu et al., 1998, Nucl. Acids Res. 26:2034-2035. Even when the methods of the present invention are used with suspension cells, the methods have the adv antage over prior art methods that the suspension cells need not be harvested before they are assayed for FRET.
  • ratiometric analysis affords much greater sensitivity to assays for caspase activity than does the use of Western blotting.
  • increased sensitivity is of great value in the use of such assays to identify activators of the expression of caspases and inhibitors of caspase activity.
  • the present invention includes a fusion protein and its use in the methods described herein where all of the amino acids in the fusion protein are derived from the ammo acids of the donor and acceptor GFPs except for a four or five amino acid linker that is a caspase cleavage site.
  • the inhibitors of caspases identified by the methods of the present invention will have a variety of uses. Such inhibitors will be useful in treating those conditions where it is beneficial to decrease the level of caspase activity or apoptosis. Such conditions include, e.g., neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease: traumatic brain injury; stroke; ischemia-reperfusion injury; graft-versus-host disease; and autoimmune disorders. It is clear that those skilled in the pharmaceutical arts view caspase inhibitors as having useful pharmacological activities.
  • An inhibitor of caspase- 1 (interleukin-l ⁇ converting enzyme) is currently in clinical trials for the treatment of inflammatory diseases (Press release, Vertex Pharmaceuticals, inc.. October 29, 1998).
  • a caspase inhibitor has been shown to reduce neuronal damage in a rabbit model of bacterial meningitis (Braun et al., 1999, Nature Medicine 5:298-302).
  • the activators of caspases identified by the methods of the present invention will likely be useful in the treatment of conditions where it is beneficial to increase the level of caspase activity or apoptosis, e.g., cancer or any disease that involves hyperplasia or neoplastic transformation, graft versus host disease.
  • a caspase activator/mducer of apoptosis is in clinical trials for use against graft versus host disease (Press release, Ariad Pharmaceuticals, May 6, 1999).
  • the present invention includes pharmaceutical compositions comprising activators and inhibitors of caspases that have been identified by the above-described methods.
  • the activators and inhibitors are generally combined with pharmaceutically acceptable carriers to form pharmaceutical compositions.
  • compositions containing activators and inhibitors and carriers can be found in Remington's Pharmaceutical Sciences.
  • a pharmaceutically acceptable composition suitable for effecti e administration such compositions will contain a therapeutically effective amount of the activators and inhibitors.
  • Therapeutic or prophylactic compositions are administered to an individual in amounts sufficient to treat or prevent conditions where caspase activity is abnormal.
  • the effective amount can vary according to a variety of factors such as the individual's condition, weight, gender, and age. Other factors include the mode of administration. Tne appropriate amount can be determined by a skilled physician.
  • Compositions can be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents can be desirable.
  • compositions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they car. also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • compositions can be administered in a single daily dose, or the total claiiv dosage can be administered in divided doses of two, three or four times daily.
  • compositions can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the dosage regimen utilizing the compositions is selected in accordance with a riety of factors including type, species, age, weight, sex and medical condition of the patient; the seventy of the condition to be treated; the route of administration, the renal, hepatic and cardiovascular function of the patient; and the particular composition thereof employed.
  • a physician of ordinary skill can readily determine and prescnbe the effective amount of the composition required to prevent, counter or arrest the progress of the condition
  • Optimal precision in achieving concentrations of composition within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the composition's availability to target sites. This inv olves a consideration of the distnbution, equihbnum, and elimination of a composition
  • caspase substrates were engineered to be encoded as inducible fusion proteins compnsed of tandem green fluorescent prote (GFP) molecules tethered bv ammo acid sequences that are recognized and cleaved by group II caspases (e.g., caspase-3) ( Figure IB) (Thornberry et al., 1997, J. Biol. Chem. 272: 17907-17911 1 A tetracychne-inducible expression system was used to circumvent potential problems with non-specific GFP-mediated toxicity due to prolonged overexpression (Gossen et al., 1994, Curr. Opin. Biotechnol. 5:516-520).
  • GFP tandem green fluorescent prote
  • WIB has dual excitation (434/452 nm) and emission peaks (476/505 nm), while Topaz has and excitation and emission maxima of 514 nm and 527 nm, respectively. In pnnciple.
  • caspase-3-mediated cleavage of these substrates should prevent fluorescence resonance energy transfer (FRET) between linked GFP proteins due to physical separation of the molecules.
  • FRET fluorescence resonance energy transfer
  • cleavage of the substrate can be quantitated as the ratio of excited fluorescence emitted by the acceptor molecule (Topaz, em: 527 nm i divided by the fluorescence emitted by the donor molecule (WIB, em: 476 nm).
  • WIB em: 476 nm
  • Figure 2A-B shows a direct correlation between the length of the linker region examined and the efficiency of FRET in transfected Hela cells overexpressing the fusion proteins. As the length of the linker separating the GFP molecules was increased, a corresponding decrease in FRET efficiency was observed. Energy transfer was more efficient in fusions containing the single (WT-lx) versus the tandem (WT-2x) DEVD (SEQ.ID.NO. :9) motif. Interestingly, inclusion of a flexible hinge region within the linker offered no particular advantage, but instead served to diminish FRET efficiency of the WT-2xL fusion protein relative to its parental form (WT-2x). EXAMPLE 2
  • caspase inhibition by zVAD-fmk was titrated in cultured Hela cells stably overexpressing the WT-lx construct. This fusion construct w as chosen for analysis because it represented a reasonable compromise between energy transfer efficiency and cleavability as a substrate ( Figure 2A-B and Figure 3 >.
  • Caspase inhibition was examined using intact adherent cells plated in a 96 well microtiter-like plate format. The fluorometer, equipped with the appropriate filter sets to detect FRET, was calibrated to read fluorescence emitted from intact cells lying on the surface of the plate.
  • tandem GFP assay as a high throughput screening methodology for identifying caspase inhibitors
  • duplicate sets of 96 well drug plates containing either single compounds or mixtures of ten compounds per well were tested The final concentration of each drug in these plates ranged between 400-600 ⁇ M
  • Pre-screen g of these plates revealed the absence of caspase inhibitors among the collection of test compounds (data not shown).
  • individual wells were supplemented with different concentrations of a known caspase inhibitor (zVAD-fmk).
  • the WIB GFP mutant was amplified by polymerase chain reaction (PCR) as an EcoRIAVlB/Afi ⁇ /Agel fragment from pRSETA-hWIB (Aurora Biosciences, San Diego. CA).
  • PCR polymerase chain reaction
  • a Kozak consensus (gccgccaccatgg; SEQ,ID.NO.:32) was engineered into the start methionine of WIB and the stop codon was deleted (Kozak, 1984, Nucleic Acids Res. 12:857-872).
  • Topaz was amplified by PCR as an Agel/Nhe TOPAZ/BamHI fragment from pRSETA-TOPAZ (Aurora Biosciences, San Diego, CA). The start methionine of Topaz was deleted.
  • WIB and Topaz were co-ligated into the EcoRI/BamHI sites of pIRESlneo (Clontech, Palo Alto, CA) as EcoRI/Agel and Agel BamHI fragments respectively to generate pIRESlneo-WT.
  • pIRESlneo-WT-lx pIRESlneo-WT-2x
  • pIRESlneo-WT-2xL were generated by ligation of synthetic oligonucleotides encoding DEVD (SEQ.ID.NO.:9) linker sequences into the AflD/Nhel sites of the parental pIRESlneo- W plasmid.
  • Hela cells (7.5xl ⁇ 5) were subcultured into 60 mm tissue culture dishes and transfected the following day with 2.5 ⁇ g of plasmid DNA using the Lipofectamine Plus reagent (Life Technologies). Cells were allowed to express the GFP fusions for a further 48 hours prior to analysis. To determine maximum possible FRET efficiency.
  • zVAD-fmk (100 ⁇ M) (Enzyme Systems Products) was included in the media during the transfection procedure and maintained for the 48 hours in order to inhibit caspase activity that is generated as a consequence of the transfection. In the Western blotting experiments, zVAD-fmk was included in the media as described above. For induction of apoptosis, cells were treated with staurosporine (1 ⁇ M) for 4 hours following the initial 48 hour expression period. EXAMPLE 7
  • Lysates corresponding to 1 mg of total protein (transient transfections) or complete lysate (stable cell lines) were diluted to 1 ml with 50 mM Tris, pH 7.5 and the fluorescence was measured using a Perkin Elmer LS 50B fluorometer.
  • the ratio of the intensity of the 527 nm emission peak (TOPAZ) over the intensity of the 476 nm emission peak (WIB) after excitation at 434 nm (WIB) provided us with a measurement of the level of FRET occurring between the two GFP chromaphores. No fluorescence was detected in Hela cells transfected with the empty pIRESlneo vector.
  • WT-lx and WT-2x coding sequences were subcloned from pIRESlneo into p DNA3.1(-) (Invitrogen, Palo Alto, CA) using existing EcoRI/BamHI sites. 35s]methionine-labeled WT-lx and WT-2x proteins were generated using a coupled T7-wheat germ in vitro transcription/translation system (Promega).
  • pcDNA3J(-) constructs (2 ⁇ g) were incubated with 8 ⁇ l of [35s]methionine ( 10 ⁇ Ci/ ⁇ l) (Amersham) and 2 ⁇ l RNAsin (40 U/ ⁇ l) (Boehringer Mannheim) in a total of 100 ⁇ l of wheat germ lysate for 45 minutes at 30°C.
  • Cleavage of radiolabeled WT-lx or WT-2x protein was performed by incubation with purified recombinant caspase-3 in a final volume of 10 ⁇ l of cleavage buffer (50 mM HEPES/KOH (pH 7.0).
  • Staurosponne and varying concentrations of zVAD-fmk were co-adm istered to the cells (1.5% final DMSO) and allow ed to incubate for 4 hours
  • the media was subsequently exchanged for PBS and the fluorescence from tact cells measured using a 96 well fluorometer (Perceptive Biosysytems Cytofluor) equipped with the appropnate filter sets (420/50ex, 490/40em. and 530/25em)

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Abstract

Substrats et analyses permettant l'identification d'activateurs et d'inhibiteurs de caspases. Lesdits substrats sont des protéines de fusion comprenant deux protéines fluorescentes vertes (GFPs) avec un peptide de liaison comportant au moins un site de clivage par caspase. La protéine de fusion intacte présente un transfert d'énergie de fluorescence par résonance (FRET) entre les GFPs. Suite au clivage par caspase du peptide de liaison, les deux GFPs se séparent et le FRET s'en trouve réduit.
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