WO1999018856A1 - Nouvelles molecules reporters fluorescentes, applications de ces molecules et dosages des caspases - Google Patents

Nouvelles molecules reporters fluorescentes, applications de ces molecules et dosages des caspases Download PDF

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Publication number
WO1999018856A1
WO1999018856A1 PCT/US1998/021231 US9821231W WO9918856A1 WO 1999018856 A1 WO1999018856 A1 WO 1999018856A1 US 9821231 W US9821231 W US 9821231W WO 9918856 A1 WO9918856 A1 WO 9918856A1
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Prior art keywords
seq
rhodamine
cells
compound
asp
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PCT/US1998/021231
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English (en)
Inventor
Eckard Weber
Sui Xiong Cai
John F. W. Keana
John A. Drewe
Han-Zhong Zhang
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Cytovia, Inc.
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Priority to CA002308125A priority Critical patent/CA2308125A1/fr
Application filed by Cytovia, Inc. filed Critical Cytovia, Inc.
Priority to BR9814816-8A priority patent/BR9814816A/pt
Priority to NZ503619A priority patent/NZ503619A/en
Priority to JP2000515498A priority patent/JP2001519368A/ja
Priority to EP98953317A priority patent/EP1026988A4/fr
Priority to AU10722/99A priority patent/AU754634B2/en
Priority to IL135365A priority patent/IL135365A0/xx
Priority to EA200000408A priority patent/EA200000408A1/ru
Priority to HU0100079A priority patent/HUP0100079A2/hu
Priority to KR1020007003886A priority patent/KR20010031056A/ko
Publication of WO1999018856A1 publication Critical patent/WO1999018856A1/fr
Priority to NO20001322A priority patent/NO20001322L/no
Priority to IS5414A priority patent/IS5414A/is

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1488—Methods for deciding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention is in the field of intracellular detection of enzymes using fluorogenic or fluorescent probes.
  • the invention relates to novel fluorescent dyes and application of these dyes for the preparation of novel fluorogenic or fluorescent peptide or amino acid derivatives which are substrates of proteases and peptidases.
  • the invention relates to novel fluorogenic or fluorescent peptide derivatives which are substrates of enzymes involved in apoptosis, such as caspases and the lymphocyte-derived serine protease Granzyme
  • the invention also relates to a process for measuring the activity of caspases and other enzymes involved in apoptosis in living or dead whole cells, cell lines or tissue samples derived from any healthy, diseased, infected or cancerous organ or tissue.
  • the invention also relates to the use of the fluorogenic or fluorescent substrates in a novel assay system for discovering or detecting inhibitors or inducers of apoptosis in compound collections or compound libraries.
  • the invention relates to the use of the fluorogenic or fluorescent substrates in determining the sensitivity of cancer cells to treatment with chemotherapeutic drugs.
  • the invention also relates to novel fluorogenic or fluorescent peptide derivatives which are substrates of exopeptidases such as aminopeptidase A and N, methionine aminopeptidase and dipeptidyl-peptidase IV, endopetidases such as calpain, proteases such as HIV proteases.
  • exopeptidases such as aminopeptidase A and N, methionine aminopeptidase and dipeptidyl-peptidase IV, endopetidases such as calpain
  • proteases such as HIV proteases.
  • HCMV protease HSV protease
  • HCV protease and adenovirus protease.
  • Organisms eliminate unwanted cells by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 2(5:59-86 (1951); Glucksmann, A., Archives de Biologie 76:419-431 (1965); Ellis et al., Dev. 772:591-603 (1991); Vaux et al., Cell 76:111-119 (1994)).
  • Apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function.
  • apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (PCT published application WO96/20721).
  • Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and
  • a cell activates its internally encoded suicide program as a result of either internal or external signals.
  • the suicide program is executed through the activation of a carefully regulated genetic program (Wylie et al, Int. Rev. Cyt. 68:251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7:663 (1991)).
  • Apoptotic cells and bodies are usually recognized and cleared by neighboring cells or macrophages before lysis. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells (Orrenius, S., J. Internal Medicine 237:529- 536 (1995)).
  • IL-1B Mammalian interleukin-l ⁇
  • IL-1B plays an important role in various pathologic processes, including chronic and acute inflammation and autoimmune diseases (Oppenheim, J. H. et. al. Immunology Today, 7, 45-56 (1986)).
  • IL-l ⁇ is synthesized as a cell associated precursor polypeptide (pro-IL-l ⁇ ) that is unable to bind IL-1 receptors and is biologically inactive (Mosley et al, J. Biol Chem.
  • IL-1 is also a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis and growth of certain leukemias (Dinarello. C.A.. Blood 77: 1627-1652 (1991 ); diGiovine et al,
  • Interleukin-l ⁇ converting enzyme is a protease responsible for the activation of interleukin-l ⁇ (IL-l ⁇ ) (Thornberry, N.A., et al, Nature 356:16 (1992); Yuan, J., et al. Cell 75:641 (1993)).
  • ICE is a substrate-specific cysteine protease that cleaves the inactive prointerleukin-1 to produce the mature IL-1.
  • the genes that encode for ICE and CPP32 are members of the mammalian ICE/Ced-3 family of genes which presently includes at least twelve members: ICE, CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2, MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICErellll.
  • This gene family has recently been named caspases (Alnernri, E. S. et.
  • a death trigger such as Tumor Necrosis Factor, FAS-ligand, oxygen or nutrient deprivation, viruses, toxins, anti-cancer drugs etc.
  • caspases upstream in the cascade e.g. FLICE/MACH/MCH5
  • capsases further downstream in the cascade e.g. CPP- 32/Yama/Apopain. Activation of the caspase cascade leads to cell death.
  • Caspases are also thought to be crucial in the development and treatment of cancer. There is mounting evidence that cancer cells, while containing caspases. lack parts of the molecular machinery that activate the caspase cascade
  • chemotherapeutic drugs can trigger cancer cells to undergo suicide by re-activating the dormant caspase cascade.
  • Chemotherapeutic drugs may differ in their capacity to activate the caspase system in different classes of cancers. Moreover, it is likely that anti-cancer drugs differ in their ability to activate the caspase cascade in a given cancer (e.g. lung cancer) and in different patients. In other words, there are differences from one patient to another in the chemosensitivity of, e.g. lung cancer cells, to various anti-cancer drugs.
  • the excessive activation of the caspase cascade plays a crucial role in a wide variety of degenerative organ diseases, while a non-functioning caspase system is a hallmark of cancer cells.
  • New drugs that inhibit or stimulate the caspase cascade are likely to revolutionize the treatment of numerous human diseases ranging from infectious, cardiovascular, endocrine, kidney, liver and brain diseases to diseases of the immune system and to cancer.
  • HTCA high-throughput caspase activation
  • HTCA assays should be versatile enough to measure the caspase cascade activity inside any living or whole cell, no matter what its origin might be: Cancer cells, tumor cells, immune cells, brain cells, cells of the endocrine system, cells or cell lines from different organ systems, biopsy samples etc. Furthermore, such HTCA assays should be able to measure—within living or whole cells—the activation or inhibition of any of the caspase enzymes or any other enzymes that are involved in the caspase cascade. Developing such versatile HTCA assays represents a substantial advance in the field of drug screening.
  • HTCA assays do not permit inner cellular screening for compounds that can either activate or inhibit the caspase cascade.
  • a potentially important application of a HTCA assay system for measuring intracellular caspase enzymes or any other enzymes involved in apoptosis is chemosensitivity testing of human cancers. It is known that there is a genetic difference in the susceptibility of human cancers to the currently marketed anti-cancer drugs: For example, lung cancer cells in one patient might be sensitive to Drug A, while another patient's lung cancer might be insensitive to Drug A, but sensitive to Drug B. This pharmacogenetic difference in chemosensitivity of cancer cells from different individuals is a well-known phenomenon.
  • chemosensitivity testing has not found wide-spread use, because the procedures involved have some inherent technical difficulties: The testing is very time consuming (six or more days per screen) and it requires culturing of the cells prior to screening. The cell culture leads to clonal selection of cells and the cultured cells are then no longer representative of the cancer in the patient.
  • a HTCA assay system for quickly measuring intracellular caspase activity could be used to determine very rapidly the chemosensitivity profile of freshly excised cancer cells.
  • the assay has a high throughput, it would be feasible to test chemosensitivity of multiple samples taken from the same patient, e.g. from different metastases. This information could then be used to design a treatment regimen using combinations of marketed anti-cancer drugs to which the cells showed greatest sensitivity.
  • HTCA assays and reagents for such assays that can be employed in drug discovery or diagnostic procedures to quickly detect and measure the activity of compounds that activate or inhibit the caspase cascade or other enzymes involved in apoptosis in the interior of living or dead whole cells.
  • a reagent for this type of cell assay ideally should meet the following conditions: a) there should be a big difference in fluorescence signal between peptide-reporter molecule and reporter molecule after the amide bond in peptide-reporter is cleaved by the caspases or other enzymes involved in apoptosis, preferably the peptide-reporter molecule should be non-fluorescent and most preferably the peptide-reporter molecule should be non-fluorescent and colorless; b) the peptide-reporter molecule should be cell permeable, therefore there should be minimum numbers of hydrophilic groups in the molecule and the size of the molecule should preferably be small; c) the peptide-reporter molecule should preferably not diffuse out of the cell once it permeates the cell membrane; d) the reporter molecule should preferably not diffuse out of the cell once it is liberated from the peptide.
  • the method of screening apoptosis inhibitors or inducers in whole cells vs cell-free enzyme assay can also be used for the screening of inhibitors of enzymes other than caspases.
  • enzyme inhibitors were first identified by cell- free enzyme assays. Cell cultures were then used for secondary assay to assess activity of the active compounds in intact cells.
  • a cell permeable fluorogenic or fluorescent substrate will enable the screening of inhibitors of proteases and peptidases and other enzymes directly in living whole cells.
  • AGM-1470 (also known as TNP-470) is an angiogenesis inhibitor in clinical trials for a variety of cancers.
  • the mechanism of action of AGM-1470 was recently discovered by two independent research groups (Sin, N., et al. Proc. Natl Acad. Sci. U.S.A. 94:6099-6103 (1997); Griffith, E.C., et al, Chem. Biol. 4:461-471 (1997)). They found that AGM-1470 and analogs are inhibitors of methionine animopeptidase type 2 (MetAP-2). The potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined for a series of AGM-1470 analogs and a significant correlation between the two activities was found.
  • angiogenesis inhibitors are known to be able to selectively kill cancer cells
  • a cellular screening assay for inhibitors of MetAP-2 may result in novel anti-cancer drugs. Therefore cell permeable fluorogenic or fluorescent substrates for MetAP-2 can be used for the screening of inhibitors of MetAP-2 in endothelial cells which could lead to novel anticancer agents.
  • HIV protease inhibitors such as ritonavir and viracept have been shown to be very effective in the treatment of patients infected with HIV. These inhibitors were designed based on the structure of the HIV protease substrate.
  • a cell permeable fluorogenic or fluorescent substrate for HIV protease can be used for the screening of HIV protease inhibitors in HIV infected cells which could speed up the process for the discovery of novel HIV protease inhibitors and lead to new and better treatment for HIV infection. Since HIV protease processes viral precursor proteins at a late stage in viral replication, a cell permeable fluorogenic or fluorescent substrate for HIV protease also can be used to screen compounds which inhibit gene transcription or translation, viral entry, or other key proteins in the early stage of HIV infection. The fluorogenic or fluorescent substrates also could be used for diagnosis of HIV infection, which might be more sensitive than the currently available methods.
  • cell permeable fluorogenic or fluorescent substrates for cathepsin B can be used for the screening of cathepsin B inhibitors.
  • Cell permeable fluorogenic or fluorescent substrates for dipeptidyl-peptidase IV can be used for the screening of dipeptidyl-peptidase IV inhibitors.
  • Cell permeable fluorogenic or fluorescent substrates for renin can be used for the screening of renin inhibitors and cell permeable fluorogenic or fluorescent substrates for adenovirus protease or other viral proteases can be used for the screening of adenovirus protease or other viral protease inhibitors.
  • U.S. Patent Nos. 4,557,862 and 4,640,893 disclose Rhodamine 1 10 derivatives as fluorogenic substrates for proteinases of the formula:
  • R, and R 2 which are the same or different, are selected from the group consisting of amino acids, amino acid derivatives, blocked amino acids, blocked amino acid derivatives, and peptides.
  • Exemplary (AA) 2 -Rhodamines and (peptide) 2 -Rhodamines are (Z-Arg) 2 -Rhodamine 1 10, (Arg) 2 -Rhodamine 1 10, (Z- Ala-Arg) 2 -Rhodamine 110, (Z-GLV-Arg) 2 -Rhodamine 110, (Z-Glu-Arg) 2 - Rhodamine 1 10, (Z-Gly-Arg) 2 -Rhodamine 1 10, (Z-Leu-Arg) 2 -Rhodamine 110, (Z-Met-Arg) 2 -Rhodamine 110, (Z-Phe-Arg) 2 -Rhodamine 1 10.
  • WO 96/36729 discloses compounds or their salts for assaying the activity of an enzyme inside a metabolically active whole cell.
  • the assay compound is said to include a leaving group and an indicator group.
  • the leaving group is selected from the group comprising amino acids, peptides, saccharides. sulfates, phosphates, esters, phosphate esters, nucleotides, polynucleotides, nucleic acids, pyrimidines, purines, nucleosides, lipids and mixtures.
  • the indicator group is selected from compounds which have a first state when joined to the leaving group, and a second state when the leaving group is cleaved from the indicator group by the enzyme.
  • Preferred indicator compounds are said to be Rhodamine 110. rhodol, and fluorescein and analogs of these compounds.
  • the patent application listed many enzymes and substrates of enzymes.
  • US patent 5,576,424 disclosed haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells of the formula: XR-SPACER-REPORTER-BLOCK
  • -BLOCK is a group selected to be removable by action of a specific analyte, to give reporter spectral properties different from those of the substrate
  • - REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK- REPORTER bond, has spectral properties different from those of the substrate
  • SPACER- is a covalent linkage; and XR- is a haloalkyl moiety that can covalently react with an intracellular thiol to form a thioether conjugate.
  • Preferred reporter compounds are said to include Rhodamine- 1 10. rhodol. fluorescein and others.
  • the invention relates to fluorogenic or fluorescent reporter compounds of Formula I: x-y-z (I) or biologically acceptable salts or pro-reporter molecules (such as methyl ester form of carboxyl-containing amino acid residues) thereof, wherein x and z is the same or different and is a peptide or amino acid or acyl group or other structure such that compounds of Formula I is a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and wherein the scissile bond is only one or both of the x-y and y-z bonds in Formula I when x is the same as z, or wherein the scissile bond is only one of the x-y or y-z bond in Formula I when x is not the same as z.
  • y is a fluorogenic or fluorescent moiety.
  • Preferred compounds are represented by Formula II:
  • R is an N- terminal protecting group such as t-butyloxycarbonyl, acyl, and benzyloxycarbonyl; each AA independently is a residue of any natural or non- natural ⁇ -amino acid or ⁇ -amino acid, or derivatives of an ⁇ -amino acid or ⁇ - amino acid; each n is independently 0-5; and y is a fluorogenic or fluorescent moiety.
  • Preferred y is a Rhodamine including Rhodamine 110, Rhodamine 116 and Rhodamine 19. Most preferred y is Rhodamine 110.
  • R is t-butyloxycarbonyl, acyl and benzyloxycarbonyl.
  • Preferred values of n are 1-3.
  • the invention also relates to a method for the preparation of a compound of Formula III, comprising
  • -(AA) n is WEH SEQ ID ⁇ O:l, YVA SEQ ID NO:2, LEH SEQ ID NO:3, DET SEQ ID NO:4, DEV SEQ ID NO:5, DEH SEQ ID NO:6, VEH SEQ ID NO:7, LET SEQ ID NO:8, LEV SEQ ID NO:9, SHV SEQ ID NO: 10, DEL SEQ ID NO:l l, DGP SEQ ID NO: 12, DEP SEQ ID NO:13, DGT SEQ ID NO:14, DLN SEQ ID NO:15, DEE SEQ ID NO:16, DSL SEQ ID NO: 17, DVP SEQ ID NO: 18, DEA SEQ ID NO: 19, DSY SEQ ID NO:20, ELP SEQ ID NO:21, VED SEQ ID NO:22, IEP SEQ ID NO:23 or IET SEQ ID NO:24, and the carboxy containing amino acids are protected with an OBu-t group which is removed in the final step.
  • Another group of preferred compounds falling within the scope of Formula I include compounds wherein x is not the same as z.
  • Preferred compounds of this group include those wherein x is a peptide or other structure which makes the compound a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and the x-y bond in Formula I is the scissile bond under biological conditions, z is a blocking group and the y-z bond in Formula I is not a scissile bond under biological conditions.
  • novel fluorogenic or fluorescent reporter compounds of this invention are of Formula V:
  • R,, AA, n and y are as defined previously in formula II;
  • R 6 is a blocking group which is not an amino acid or a derivative of an amino acid.
  • R 2 and R are the same or different and are independently hydrogen, alkyl or aryl
  • R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • R,, R 6 , AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
  • R 2 and R are the same or different and are independently hydrogen, alkyl or aryl
  • R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • R ] , R 6 , AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
  • R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl; and R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • the invention also relates to a method for the preparation of a compound of Formula VII, comprising
  • the invention also relates to the novel fluorescent dyes of Formula VI which are derivatives of Rhodamines.
  • These compounds are prepared by introducing a blocking group R ⁇ into one of the two amino groups of Rhodamine.
  • the R 2 HN group in Formula VI provides the point of attachment for reaction with a potential enzyme substrate, such as the carboxylic group of a N-blocked peptide, to form a peptide amide bond.
  • the reaction will convert the fluorescent molecule of Formula VI into a non-fluorescent peptide-reporter molecule of Formulae VII- IX which is a substrate for a protease or peptidase. Cleavage of the scissile peptide-reporter amide bond in peptide-reporter by proteases or peptidases produces compound of Formula VI or VI' which is fluorescent.
  • novel fluorescent dyes of this invention are of Formula VI:
  • R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl;
  • R is a blocking group which is not an amino acid or a derivative of an amino acid
  • R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • Preferred R 2 and R 3 are hydrogen, methyl or ethyl; Preferred R 4 and R 5 are hydrogen or methyl.
  • the invention also relates to a process of using the reporter compounds represented by Formula I to measure the activity of intracellular caspases or other enzymes involved in apoptosis in living or dead whole cells or tissues.
  • the invention also relates to methods of using the compounds represented by Formula I and the assay processes described herein to measure the activation or inhibition of any of the caspase enzymes inside any living or dead whole cell or tissue (normal or cancerous) by a test substance or substances.
  • the compounds represented by Formula I are cell-permeable, that is, they can be introduced into whole cells or tissue samples.
  • the compounds are fluorogenic or fluorescent and can be designed to be specific for any of the known caspases or for any other intracellular enzymes involved in apoptosis.
  • fluorogenic or fluorescent substrates for specific caspases can be synthesized by the procedures described herein.
  • the fluorogenic or fluorescent substrates can also be designed to measure more than one enzyme at a time, by designing substrates that are recognized and cleaved by more than one of the enzymes involved in the caspase cascade. Fluorogenic or fluorescent substrates which are "promiscuous" for more than one caspase may be utilized using the assay process described herein to measure the activity of as yet unknown caspases.
  • the fluorogenic or fluorescent reporter molecules described herein are cleaved and respond with a large increase in fluorescence emission.
  • the change in fluorescence can be measured spectrofluorometrivally.
  • the reporter molecules can also be used to measure baseline caspase activity in cells that are not undergoing apoptosis.
  • the method is easily adaptable to high throughput or ultra- high throughput screening assays.
  • the assay system is very versatile. Examples of the extreme versatility of the assay system are given below:
  • the assay can be used to screen a cell or tissue for baseline activity of any caspase enzyme or any other enzyme involved in apoptosis. 2.
  • the assay can be used with equal ease to screen for compounds that can either activate or inhibit the caspase cascade. That means the assay can be used to screen for drugs against degenerative diseases or for drugs against cancer.
  • the assay can be used to screen for caspase cascade activation or inhibition in any living or dead cells or cell lines derived from any organ system in the body including, but not limited to. hair, brain, peripheral nervous system, eye, ear. nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands, joints, bones and skin.
  • the assay can be used to screen for drugs with potential use in any disease of any organ system in the body that involves malfunction of the caspase cascade.
  • the assay can be used to screen for drugs that might modulate the caspase cascade directly or indirectly, i.e. by modulating the caspases itself or by modulating cellular receptors and co-factors that influence the caspase cascade.
  • the assay can be used to determine the site of action at which a caspase cascade modulator interferes. That is, the assay can help to pin down the molecular mechanism of action of a novel caspase cascade modulator drug.
  • the invention also relates to the use of the fluorogenic or fluorescent substrates represented by Formula I for finding new compounds or new uses for known compounds in reducing, preventing or treating maladies in which apoptotic cell death is either a causative factor or a result.
  • uses for the present invention include screening for compounds that can protect the nervous system following focal ischemia and global ischemia: screening for compounds that can treat neurodegenerative disorders such as Alzheimer's disease, Huntington' s Disease, prion diseases, Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, telangiectasia, and spinobulbar atrophy; screening for compounds that can treat heart disease including myocardial infarction, congestive heart failure and cardiomyopathy; screening for compounds that can treat retinal disorders; screening for compounds that treat autoimmune disorders including lupus erythematosus, rheumatoid arthritis, type I diabetes, Sj ⁇ gren's syndrome and glomerulonephritis; screening for compounds that
  • the present invention also relates to the use of the fluorogenic or fluorescent substrates represented by Formula I in screening procedures where libraries of known drugs or combinatorial or other compound libraries are screened for compounds with anti-tumor or anti-cancer activity.
  • the cancer cells or cell lines can be derived from any cancer of any internal or external organ system in the body including, but not limited to brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands (e.g. prostate gland), joints, bones and skin.
  • brain peripheral nervous system
  • eye ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal
  • the present invention also relates to the use of the fluorogenic or fluorescent substrates represented by Formula I in diagnostic procedures to determine the chemosensitivity or resistance of cancer cells taken from an animal or a human being to treatment with chemotherapeutic drugs.
  • the cancer cells or cell lines can be derived from any cancer of any internal or external organ system in the body including, but not limited to brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands (e.g. prostate gland), joints, bones and skin.
  • brain peripheral nervous system
  • eye ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal
  • the invention relates to a method for detecting an enzyme involved in the apoptosis cascade in one or more cells, comprising (a) contacting the one or more cells with a reporter compound according to the invention under conditions whereby the reporter compound is taken into said one or more cells, and
  • the invention also relates to a method for measuring the activity of an enzyme involved in the apoptosis cascade in one or more cells, comprising
  • the invention also relates to a method for determining whether a test substance has an effect on an enzyme involved in the apoptosis cascade in one or more test cells, comprising
  • test cells may be contacted with said test substance prior to, after, or substantially simultaneously with the reporter compound according to the invention.
  • the method may be used to detect whether the test substance stimulates or inhibits the activity of the enzyme.
  • the invention also relates to further contacting the test cells with a second test substance or mixture of test substances in the presence of the first test substance.
  • the test cell is a cancer cell or cell line derived from a human in need of treatment with a chemotherapeutic drug and the test substance is a chemotherapeutic agent or a mixture of chemotherapeutic agents.
  • the invention also relates to a method to determine the sensitivity of an animal with cancer to treatment with one or more chemotherapeutic agents, comprising
  • the invention also relates to a method to monitor the treatment of an animal with one or more chemotherapeutic drugs, comprising (a) administering one or more chemotherapeutic drugs to the animal,
  • the animal may suffer from a malady in which apoptotic cell death is either a causative factor or a result.
  • the invention also relates to a method for determining whether a test substance inhibits or prevents cell death in one or more test cells, comprising
  • test substance either interacts with an external membrane receptor or is taken into the cell and the reporter compound is taken into the cell
  • the invention also relates to a method for determining whether a test substance causes or enhances cell death in one or more test cells, comprising
  • test substance (a) contacting the test cells with the test substance and the reporter compound according to the invention under conditions whereby the test substance either interacts with an external membrane receptor or is taken into the cells and the reporter compound is taken into the cells,
  • the invention also relates to a process of using the reporter compounds represented by Formula IX to measure the activity of intracellular peptidases and proteases in living whole cells, including, but not limited to, type-2 methionine aminopeptidase in endothelial cells and HIV protease in HIV infected cells.
  • the invention also relates to methods of using the compounds represented by Formula IX to measure the activity of intracellular peptidases and proteases in living whole cells, including, but not limited to, type-2 methionine aminopeptidase in endothelial cells and HIV protease in HIV infected cells.
  • the invention also relates to methods of using the compounds represented by Formula
  • the reporter compounds represented by Formula IX are cell-permeable, that is they can be introduced into whole cells.
  • the compounds are fluorogenic or fluorescent and can be designed to be specific for the known enzymes of interest, such as methionine aminopeptidase or HIV protease.
  • the invention also relates to a method for detecting a viral protease in one or more cells, comprising
  • the invention also relates to a method for measuring the activity of a viral protease in one or more viral infected cells, comprising
  • the invention also relates to a method for determining whether a test substance has an effect on the activity of viral protease in one or more viral infected cells, comprising
  • the cells are HIV infected cells and the viral protease is HIV protease.
  • the cells are adenovirus infected cells and the viral protease is adenovirus protease.
  • the cells are HSV infected cells and the viral protease is
  • the cells are HCMV infected cells and the viral protease is HCMV protease. In another preferred embodiment, the cells are HCV infected cells and the viral protease is HCV protease.
  • the invention also relates to a method for measuring the activity of protease or peptidase in cells, comprising (a) contacting the test cells with the reporter compound according to the invention under conditions whereby the reporter compound is taken into the test cells, or the reporter compound is interacting with an external membrane protease or peptidase of said cells, and (b) recording the fluorescence of the cells, wherein a change or increase in fluorescence within the test cell compared to control cells which have not been so contacted is a measure of the activity of the the protease or peptidase.
  • the invention also relates to a method for determining whether a test substance has an effect on the activity of protease or peptidase in the test cells, comprising
  • the cells are endothelial cells and the peptidase is type 2 methionine aminopeptidase.
  • cells are T cells and the peptidase is dipeptidyl peptidase-IV.
  • the cells are neuron cells and the protease is calpain.
  • Figs. 1A-1F depict photographs of HL-60 cells stained by N- octyloxycarbonyl-Rhodamine 110 (Fig. 1A), N-decyloxycarbonyl-Rhodamine- 1 10 (Fig. IB), N-dodecyloxycarbonyl-Rhodamine-1 10 (Fig. 1C), N- hexyloxycarbonyl-Rhodamine-110 (Fig. ID), N-(ethylthio)carbonyl-Rhodamine 1 10 (Fig. IE) and Rhodamine 110 (Fig. IF).
  • Figures 2A-2L depict the bar graphs of cleavage of the caspase substrates N-Z-VD-N'-ethoxycarbonyl-Rl 10, N-Z-EVD-N'-ethoxycarbonyl-Rl 10, N-Z- DEVD-N'-ethoxycarbonyl-Rl 10 SEQ ID ⁇ O:5, N-Ac-DEVD-N'-ethoxycarbonyl-
  • R110 SEQ ID ⁇ O:5, N-Ac-DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5, N- Ac-DEVD-N'-hexyloxycarbonyl-Rl lO SEQ ID NO: 5. and N-Z-DEVD-N- (ethylthio)carbonyl-RHO SEQ ID ⁇ O:5, by r-caspase-3 (Figs. 2A, 2B,_ 2D, 2G and 2J) and Vinblastine treated HL-60 cell lysates (Figs. 2C, 2E, 2H and 2K) compared to HL-60 control (DMSO treated) lysates (Figs. 2F, 21 and 2L).
  • Figs. 3A-3E depict photographs of cells stained by incubation with N-Ac-
  • DMSO (Fig. 3B) treated HL-60 cells, vinblastine treated HL-60 cells with N-Ac-
  • Fig. 4 depicts a graph showing the results of a cleavage assay of N-Ac- DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5 by antiFas and PBS treated Jurkat cells.
  • Fig. 5 depicts a bar graph showing the results of a cleavage assay of N-Ac- LEVD-N'-ethoxycarbonyl-Rl 10 SEQ ID ⁇ O:5 by caspase-3, -6, -7 and -8.
  • Fig. 6 depicts a bar graph with the results of a cleavage assay of N-Z-G-N- octyloxycarbonyl-RHO and N-G-N'-octyloxycarbonyl-Rl 10 by HL-60 cell lysates.
  • Figures 7A-B depict photographs of HL-60 cells treated with ⁇ -Z-G- ⁇ '- octyloxycarbonyl-Rl 10 (A) and ⁇ -G- ⁇ '-octyloxycarbonyl-Rl 10 (B).
  • the fluorogenic or fluorescent substrates of the present invention are compounds having the general Formula I: x-y-z (I) or biologically acceptable salts or pro-reporter molecules (such as methyl ester form of carboxyl-containing amino acid residues) thereof, wherein x and z is the same or different and is a peptide or amino acid or acyl group or other structure such that Formula I is a substrate for caspases, or other proteases or peptidases or other enzymes; and wherein the scissile bond is only one or both of the x-y and y- z bonds in Formula I when x is the same as z, or wherein the scissile bond is only one of the x-y or y-z bonds in Formula I when x is not the same as z.
  • y is a fluorogenic or fluorescent moiety.
  • Preferred compounds falling within the scope of Formula I include compounds wherein x is the same as z. and the first amino acid attached to y is an
  • x is the same as z and is a N-blocked tetrapeptide substrate of a caspase including WEHD SEQ ID ⁇ O: l, YVAD SEQ ID NO:2, LEHD SEQ ID NO:3.
  • DGTD SEQ ID NO: 14 DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO:20, ELPD SEQ ID NO:21, VEID SEQ ID NO:26, IETD SEQ ID NO:24 or a N-blocked tetrapeptide substrate of granzyme B including IEPD SEQ ID NO: 23 and VEPD SEQ ID NO:27; or x is the same as z and is a N-blocked peptide which corresponds to a carboxyterminal or aminoterminal fragment consisting of 1 , 2 or 3 amino acids of the tetrapeptide substrate of a caspase including WEHD SEQ ID ⁇ O:l, YVAD SEQ ID NO:2, LEHD SEQ ID NO:3, DETD SEQ ID NO:4, DEVD SEQ ID NO:5, DEHD SEQ ID NO:6, VEHD SEQ
  • DGPD SEQ ID NO: 12 DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO:15, DEED SEQ ID NO:16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO:20, ELPD SEQ ID NO:21, VEID SEQ ID NO:26, IETD SEQ ID NO:24 and granzyme B including IEPD SEQ ID NO:23 and VEPD SEQ ID NO:27.
  • Preferred compounds falling within the scope of Formula I include compounds wherein y is Rhodamine 110.
  • pro-reporter molecules such as methyl ester form of carboxyl-containing amino acid residues
  • R is an ⁇ - terminal protecting group including t-butyloxycarbonyl. acetyl. benzyloxycarbonyl
  • each AA independently is a residue of any natural or non- natural ⁇ -amino acid or ⁇ -amino acid, or derivatives of an ⁇ -amino acid or ⁇ - amino acid: each n independently is 0-5; and y is a fluorogenic or fluorescent moiety.
  • An example of a pro-reporter molecule is the methyl ester form of carboxyl-containing amino acid residues comprising compounds of Formula II.
  • AM esters of carboxyl-containing compounds are known to be cell permeable and can be hydrolyzed by esterases inside the cells. Once hydrolyzed, the carboxyl- containing compounds become cell impermeable and are trapped inside the cells
  • AM esters can be prepared by reacting the corresponding carboxy-containing compounds with bromomethyl acetate.
  • R, AA, n are as defined previously in Formula II.
  • R is t-butyloxycarbonyl, acetyl and benzyloxycarbonyl. Also preferred values for n are 1 -3.
  • Another group of preferred compounds falling within the scope of Formula I include compounds wherein x is not the same as z.
  • Preferred compounds of this group include those wherein x is a peptide or other structure which makes the compound a substrate for caspases, or other proteases or peptidases or other enzymes; and the x-y bond in Formula I is the scissile bond under biological conditions; z is a blocking group and the y-z bond in Formula I is not a scissile bond under biological conditions.
  • x is a N-blocked tetrapeptide substrate of a caspase including WEHD SEQ ID ⁇ O: l, YVAD SEQ ID NO:2, LEHD SEQ ID NO:3, DETD SEQ ID NO:4, DEVD SEQ ID NO:5, DEHD SEQ ID NO:6, VEHD SEQ ID NOJ, LETD SEQ ID NO:8, LEHD SEQ ID NO:3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: l 1 , DGPD SEQ ID NO:12.
  • DEPD SEQ ID NO: 13 DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO:20, ELPD SEQ ID NO:21, VEID SEQ ID NO:26, IETD SEQ ID NO: 24 or a N-blocked tetrapeptide substrate of granzyme B including IEPD SEQ ID ⁇ O:23 and VEPD SEQ ID NO:27; or x is a N-blocked peptide which corresponds to a carboxyterminal or aminoterminal fragment consisting of 1.
  • novel fluorogenic or fluorescent reporter compounds of this invention are of Formula V:
  • R is an N-terminal protecting group including t-butyloxycarbonyl, acetyl, octanoyl and benzyloxycarbonyl; each AA independently is a residue of any natural or non-natural ⁇ -amino acid or ⁇ -amino acid, or a derivative of an ⁇ -amino acid or ⁇ -amino acid; n is 0-5; y is a fluorogenic or fluorescent moiety; and R ⁇ is a blocking group which is not an amino acid or a derivative of an amino acid.
  • novel fluorogenic or fluorescent reporter molecules of this invention of Formula VII-IX are derivatives of Rhodamines including Rhodamine 110, Rhodamine 116 and Rhodamine 19.
  • These novel fluorogenic or fluorescent reporter molecules are prepared by first introducing a blocking group R,; into one of the two amino groups of a Rhodamine to give novel fluorescent dyes of the Formula VI.
  • the remaining HNR 2 group is used for reaction with a potential enzyme substrate to give a fluorogenic substrate of Formula VII-IX.
  • By blocking one of the two amino groups in a Rhodamine the overall size of the substrate is reduced compared to a bis-substituted Rhodamine, such as a bis- peptide-Rhodamine.
  • the blocking group is selected such that a) it is stable and will not hydrolyze under biological conditions, thus amino acids are excluded because the peptide bond formed can potentially be cleaved by peptidases which are present in the cells; b) it is preferably not too bulky (e.g. is small) in order to reduce the overall size of the peptide-reporter molecule so that it will be a better enzyme substrate; c) it is preferrably hydrophobic in nature so as to increase the cellular permeability of the fluorogenic or fluorescent reporter molecule.
  • Preferred R 6 blocking groups include, but are not limited to. an C 2 ..
  • alkyloxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl and dodecyloxycarbonyl; a C 2.12 (alkylthio)carbonyl group such as (ethylthio)carbonyl, (hexylthio)carbonyl, (octylthio)carbonyl; an arylalkyloxycarbonyl group such as benzyloxycarbonyl, a C 2 .
  • acyl (alkanoyl) group such as acetyl and octanoyl, a carbamyl group such as dimethylcarbamyl, N-methyl-N-hexylcarbamyl. and an alkyl, haloalkyl or aralkyl sulfonyl group such as methanesulfonyl.
  • R 6 blocking groups are the ones that contain a hydrophobic group similar to membrane lipid, thus increasing the cellular permeability of the fluorogenic or fluorescent reporter molecules, as well as retention of the fluorescent moiety in the cells after the cleavage of substrate by targeted protease or peptidase.
  • novel fluorogenic or fluorescent reporter molecules of Formula VII- IX are prepared by reacting the amino group NHR 2 of the novel fluorescent dyes of Formula VI with a potential enzyme substrate, such as the carboxylic group of a N-blocked peptide, to form an peptide amide bond.
  • the reaction converts the fluorescent molecule of Formula VI into a non-fluorescent peptide-repoter molecule of Formulae VII-IX which is a substrate for a protease or peptidase. It is therefore very important that the blocking group R 6 - ⁇ bond of Formula VII should not be cleaved and that the peptide-reporter amide bond should be the scissile bond under biological conditions. Cleavage of the scissile peptide- reporter amide bond of Formulae VII-IX by proteases or peptidases produces a compound of Formula VI or VI' which is fluorescent.
  • Formula VII or biologically acceptable salts or pro-reporter molecules (such as methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl; R 6 is a blocking group which is not an amino acid or a derivative of an amino acid;
  • R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • R is an N-terminal protecting group; each AA independently is a residue of any natural or non-natural ⁇ -amino acid or ⁇ -amino acid, or a derivative of an ⁇ -amino acid or ⁇ -amino acid; n is 0-5; and the scissile bond is the Asp- ⁇ bond in Formula VII.
  • R 2 and R 3 are hydrogen, methyl or ethyl
  • Preferred R 4 and R 5 are hydrogen or methyl.
  • Preferred amino acids include the natural amino acids including tyrosine, glycine, phenylalanine, methionine. alanine. serine, isoleucine, leucine, threonine, valine, proline, lysine, histidine, glutamine, glutamic acid, tryptophan, arginine, aspartic acid, asparagine, and cysteine.
  • ⁇ on-natural amino acids include t- butylglycine and ⁇ , ⁇ -dimethylglutamine.
  • pro-reporter molecule is the methyl ester form of carboxyl-containing amino acid residues comprising compounds of Formula VII.
  • Another example of a pro-reporter molecule is the acetoxymethyl (AM) ester form of carboxyl-containing amino acid residues of compounds of Formula VII.
  • R,, R 6 , AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
  • R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl;
  • R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • Compounds of Formula VIII are novel fluorogenic or fluorescent substrates for caspases or other enzymes related with apoptosis.
  • cleavage of the amide bond between Asp and Rhodamine will convert the fluorogenic substrate into the fluorescent dye of Formula VI.
  • cleavage of the amide bond between Asp and (AA) m will leave the Rhodamine attached to NH 2 -(AA) m .
  • the remaining amino acids (AA) may be designed to correspond with the P' sequence of the cleavage site of substrates of caspases or apoptosis related enzymes.
  • pro-reporter molecule is the methyl or ethyl ester forms of carboxyl-containing amino acid residues comprising compounds of Formula VIII.
  • Another example of a pro-reporter molecule is the acetoxymethyl (AM) or pivaloyloxymethyl (PM) ester form of carboxyl-containing amino acid residues of compounds of Formula VIII.
  • AM esters of carboxyl-containing compounds are known to be cell permeable and can be hydrolyzed by esterases inside the cells. Once hydrolyzed, the carboxyl-containing compounds become cell impermeable and are trapped inside the cells (Adams et al, J. Am. Chem. Soc. 117:7957-7968 (1989)).
  • AM esters can be prepared by reacting the corresponding carboxy- containing compounds with bromomethyl acetate.
  • R] Re, AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
  • R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl;
  • R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • Preferred R is t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl and benzyloxycarbonyl.
  • Preferred n is 1-4.
  • Preferred R 2 and R 3 are hydrogen, methyl or ethyl.
  • Preferred R 4 and R 5 are hydrogen or methyl.
  • Preferred R ⁇ blocking groups include, but are not limited to, an C 2 _ 12 alkyloxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl and dodecyloxycarbonyl; a C 2.12 (alkylthio)carbonyl group such as (ethylthio)carbonyl, (hexylthio)carbonyl, (octylthio)carbonyl; an arylalkyloxycarbonyl group such as benzyloxycarbonyl; a C 2 _ 12 acyl (alkanoyl) group such as acetyl and octanoyl; a carbamyl group such as dimethylcarbamyl, N-methyl-N-hexylcarbamyl; and an alkyl, haloalkyl or aralkyl sulfonyl
  • (AA) is designed to be an amino acid or a peptide which is recognized by a specific peptidase or protease as the sequence in the p side and will be cleaved by the targeted peptidase or protease.
  • (AA) m is designed to be an amino acid or peptide which is recognized by a specific peptidase or protease as the sequence in the P' side, and which can be removed by aminopeptidases presented in the cells.
  • compounds of Formula IX are substrates for endopeptidases such as cathepsin D or protease such as HIV protease: when R, is H, compounds of Formula IX are substrates for exopeptidases such as methionine aminopeptidase.
  • compounds of Formula IX are designed to be substrates of type 2 methionine aminopeptidase (MetAP-2). MetAP-2 was identified recently by two research groups (Griffith, E.C., et al, Chem. Biol 4:461-471 (1997) and Sin, ⁇ ., et al, Proc.
  • MetAP-2 is a bifunctional enzyme which also regulate protein synthesis by affecting the phosphorylaton state of eIF-2.
  • AGM- 1470 is reported to only inhibit the aminopeptidase activity of MetAP-2 and have no effect on the regulatory activity of MetAP-2 (Griffith, E.C., et al, Chem. Biol. 4:461-471 (1997)). Since angiogenesis inhibitor such as AGM-1470 is known to be able to selectively kill cancer cells, inhibitors of MetAP-2 are expected to have anti-angiogenic properties and to be potential novel anticancer agents.
  • MetAP-2 is a cobalt-dependent enzyme that hydrolyzes the aminoterminal methionine from certain proteins. Its preferred substrates are Met-X-Y.
  • X is an amino acid with small and uncharged side groups, such as Gly, Ala, Ser, whereas Leu, Met, Arg and Tyr are known to result in inactive substrates.
  • Y can be Ser, Met, Gly or other amino acids (Li, X. & Chang Y.-H., Biochem. Biophy. Res. Com. 227. 152-159 (1996)). Since Rhodamine is much larger than an amino acid, a compound with methionine directly attached to Rhodamine most probably will not be a substrate for MetAP-2.
  • a (AA) m sequence between methionine and Rhodamine will make a good substrate for MetAP-2.
  • This type of substrate is expected to work well in a whole cell assay but otherwise will not work in a cell-free MetAP-2 enzyme assay.
  • preferred R is H
  • preferred (AA) n is Met
  • preferred (AA) m is Gly, Ala, Gly- Gly, Ala-Gly or Gly-Ala.
  • the methionine will be cleaved by type 2 methionine aminopeptidase in endothelial cells to give the Rhodamine attached to (AA) m . Aminopeptidases present inside the cells will then remove the (AA) m to give the fluorescent dye of Formula VI.
  • Compounds of Formula IX will be used for the screening of inhibitors of MetAP-2 in endothelial cells, which is expected to lead to the identification of novel anti-cancer drugs.
  • HIV protease is an aspartic protease which processes polypeptides transcribed from the gag and pol genes and is essential for the maturation of infectious virus. Therefore HIV protease has been one of the major targets for chemotherapeutic intervention of HIV.
  • HIV protease inhibitors have shown great potential in the treatment of HIV and have been approved for marketing. Most of these HIV protease inhibitors were designed based on the structure of the substrates of the protease. Therefore these compounds are either peptides or peptidomimetics. The search for new and novel HIV protease inhibitors is expected to provide more efficacious drugs for the fight against this deadly disease.
  • the preferred substrates of HIV protease are peptides with a scissile hydrophobic-hydrophobic or aromatic-proline peptide bond between the P P)'
  • Gln-Arg SEQ ID NO:30 Arg-Gln-Ala-Asn-Phe-Leu-Gly SEQ ID NO:31, Pro- Gly-Asn-Phe-Leu-Gln-Ser SEQ ID NO:32, Ser-Phe-Ser-Phe-Pro-Gln-Ile SEQ ID NO:33, Thr-Leu-Asn-Phe-Pro-Ile-Ser SEQ ID NO:34, Ala-Glu-Thr-Phe-Tyr-Val- Asp SEQ ID NO:35 and Arg-Lys-Val-Leu-Phe-Leu-Asp SEQ ID NO:36.
  • HIV protease substrates have been developed, and these include the fluorogenic N-alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta-naphthylamide SEQ ID ⁇ O:37, which contains the Phe-Pro dipeptide bond recognized by HIV-1 protease (Tyagi, S.C., and Carter, C.A., Anal. Biochem.
  • preferred R is acetyl or Cbz
  • preferred (AA) n is Thr-Ile-Nle
  • preferred (AA) ra is Phe-Gln-Arg, Phe-Gln, or Phe
  • preferred (AA) n is Ser-Leu-Asn-Phe SEQ ID NO: 1
  • Glu-Ala, Phe-Glu, or Phe; or preferred (AA) peer is Pro-Phe-His-Leu SEQ ID NO:50, or Phe-His-Leu
  • preferred (AA) m is Leu-Glu-Glu-Ser SEQ ID NO:40, Leu- Glu-Glu, Leu-Glu, or Leu
  • preferred (AA) ⁇ is Ser-Gln-Asn-Leu-Phe SEQ ID NO: 140, Gln-Asn-Leu-Phe SEQ ID NO:51, Asn-Leu-Phe, Arg-Lys-Ile-Leu-Phe SEQ ID NO: 52, Lys-Ile-Leu-Phe SEQ ID NO: 53, or Ile-Leu-Phe, and preferred
  • (AA) m is Leu-Asp-Gly-NH 2 , Leu-Asp-NH 2 . or Leu-NH 2 . More preferred (AA) thread is Ser-Leu-Asn-Phe SEQ ID NO: 54, or Leu-Asn-Phe, and more preferred (AA), situation is Pro-Ile-Val, Pro-He, or Pro; or more preferred (AA) thread is Arg-Gly-Phe, and more preferred (AA) m is Pro. Substrates of HIV protease of Formula IX are expected to work in whole cell assays but otherwise will not work in cell-free enzyme assays.
  • HIV protease processes viral precursor proteins at a late stage in viral replication
  • a cell permeable fluorogenic or fluorescent substrate for an HIV protease also can be used to screen compounds which inhibit gene transcription or translation, viral entry, or other key proteins in the early stage of HIV infection. Therefore this method can lead to the identification of inhibitors of HIV infections with a novel mechanism, which could not be identified in a cell-free enzyme assay.
  • substrates of Formula V also can be used for the diagnosis of HIV infection.
  • Compounds of Formula IX also can be designed to be substrates of adenovirus protease.
  • Adenovirus are the cause of several diseases including sporatic respiratory disease and epidemic acute respiratory disease which can lead to preumonia.
  • Adenovirus protease is a cysteine protease which cleaves several viral proteins and is required for virus maturation and infectivity (Weber, J.M., Curr. Top. Microbiol Immunol. 199/1:221-235 (1995)).
  • the preferred substrates of adenovirus protease includes (M.LJ)XGX-G and (M,L,I)XGG-X.
  • the specificity of the substrates are mainly determined by P 2 and P 4 amino acids (Diouri, M., et al, J. Biol. Chem. 277:32511-32514 (1996)). Hydrophobic amino acids such as Met, Leu and He are perferred in P 4 . Small amino acid such as Gly is preferred in P 2 . A small and hydrophobic amino acid is also preferred for P, and P,', such as Ala and Gly; while P 3 can accommodate almost any amino acid.
  • fluorogenic or fluorescent substrates of adevovirus protease can be designed to incorporate amino acids either from the P side only, or from both the P side and P' side of adenovirus protease substrate for application in whole cell assays.
  • preferred R is acetyl or Cbz
  • preferred (AA) conflict is Leu-Arg-Gly-Gly SEQ ID NO:55, Met-Arg-Gly-Gly SEQ ID NO:56, Ile-Arg-Gly-Gly SEQ ID NO:57,
  • Compounds of Formula IX will be used for the screening of inhibitors of adenovirus protease in adenovirus infected cells.
  • Compounds of Formula IX also can be designed to be substrates of herpes simplex virus type 1 (HSV-1) protease.
  • HSV-1 protease Human herpes simplex virus type 1 is responsible for herpes labialis (cold sores).
  • the HSV-1 protease is a serine protease and is responsible for proteolytic processing of itself and ICP35 for assembly of viral capside (Gao, M, et al, J. Virol 68:3102-3112 (1994)).
  • Two proteolytic sites have been identified to be Ala247 and Ser248 and Ala610 and
  • HSV-1 protease resides within the P ⁇ P region of the cleavage sites (McCann, P.J., et al, J. Virol. (55:526-529 (1994)).
  • fluorogenic or fluorescent substrates of HSV-1 protease are designed to incorporate amino acids either from the P 4 -P, only, or both from P 4 -P, and P' side of HSV-1 protease substrate for application in whole cell assays.
  • preferred R is acetyl or Cbz
  • preferred (AA) n is Leu-Val-Leu-Ala SEQ ID NO:62
  • Compounds of Formula IX will be used for the screening of inhibitors of HSV-1 protease in HSV-1 infected cells.
  • Compounds of Formula IX also can be designed to be substrates of human cytomegalovirus (HCMV) protease.
  • HCMV can cause life-threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer or transplant patients.
  • Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein and is essential for virus replication, therefore it is a potential target for therapeutic intervention.
  • the HCMV protease is a serine protease and two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). (Sztevens, J.T., et al, Eur. J. Biochem. 226:361-361 (1994)).
  • Val-Asn-Ala-Ser-Ser-Arg-Leu-Ala-EDANS SEQ ID NO:63 was synthesized and found to be cleaved efficiently by CMV protease at the Ala-Ser peptide bond (Holskin, B.P., et al, Anal. Biochem. 227:148-155 (1995)). Recently, it was reported that replacement of the Val-Val-Asn sequence corresponding to the P 4 -P 2 residues of the maturation site of the enzyme by the optimized Tbg-Tbg-
  • Asn(NMe 2 ) (Tbg, t-butylglycine) sequence increase significant the affinity of the substrate to the protease.
  • An AMC fluorogenic substrate was prepared with the P side peptide sequence including these improved amino acids (Bonneau, P.R., et al, Anal. Biochem. 255:59-65 (1998)).
  • fluorogenic or fluorescent substrates of HCMV protease are designed to incorporate amino acids either from the P side only, or both from P side and P' side of HCMV protease substrate for application in whole cell assays.
  • R is acetyl or Cbz
  • preferred (AA) n is Val-Val-Asn-Ala SEQ
  • HCV hepatitis C virus
  • HCV hepatitis C virus
  • HCV protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein, thus the NS3/4A protease complex is an attractive target for antiviral therapy against HCV.
  • the HCV protease is a serine protease and Cys-Ser has been identified as a cleavage site.
  • One of the substrate sequence is Asp-Asp-Ile-Val-Pro-Cys-Ser- Met-Ser-Tyr SEQ ID NO:66, and P, Cys and P 3 Val were found to be critical (Zhang, R., et al, J. Virol. 77:6208-6213 (1997)).
  • fluorogenic or fluorescent substrates of HCV protease are designed to incorporate amino acids both from the P side and
  • preferred R is acetyl or Cbz
  • preferred (AA) is Asp-Asp-Ile-Val-Pro-Cys SEQ ID NO:67, Asp-Ile-Val-Pro-Cys SEQ ID NO:68, or Ile-Val-Pro-Cys SEQ ID NO:69
  • preferred (AA) m is Ser-Met-Ser-Tyr SEQ ID NOJ0, Ser-Met-Ser, Ser-
  • Formula IX will be used for the screening of inhibitors of HCV protease in HCV infected cells.
  • the invention also relates to novel compounds of Formula VI which are derivatives of a Rhodamine and are obtained by introducing a blocking group R 6 onto one of the two amino groups on a Rhodamine.
  • the R 2 HN group in Formula VI provides the point of attachment for the reaction with a potential enzyme substrate, such as the carboxylic group of a N-blocked peptide, to form an peptide amide bond.
  • the reaction converts the fluorescent molecule of Formula VI into a non-fluorescent molecule of Formulae VII-IX and produces a peptide-reporter molecule which functions as a substrate for a protease or peptidase.
  • the peptide- reporter amide bond in Formulae VII-IX is the scissile bond under biological conditions.
  • the blocking group can incorporate a hydrophobic group.
  • the hydrophobic group is designed to increase the membrane permeability of the substrates, and to result in an accumulation of the substrate inside the cells, as well as to increase retention of the fluorescence moiety inside the cells after its cleavage by targeted protease or peptidase.
  • novel fluorescent dyes of this invention are of Formula VI:
  • R 2 and R 3 are hydrogen, methyl or ethyl
  • Preferred R 4 and R 5 are hydrogen or methyl.
  • Compounds of Formula VI of the present invention may exist in tautomeric forms, particularly the ring opening form of Formula VI'.
  • the invention includes all tautomeric forms including VI and VI'.
  • Preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula II and include, but are not limited to: (Z-WEHD) 2 -Rhodamine 110, SEQ ID NO:l (Z-YVAD) 2 -Rhodamine 110, SEQ ID NO:2 (Z-DETD) 2 -Rhodamine 110, SEQ ID NO:4 (Z-DEVD) 2 -Rhodamine 1 10, SEQ ID NO:5 (Z-DEHD) 2 -Rhodamine 110, SEQ ID NO:6 (Z-VEHD) 2 -Rhodamine 1 10, SEQ ID NOJ (Z-LETD) 2 -Rhodamine 1 10, SEQ ID NO: 8 (Z-LEHD) 2 -Rhodamine 110, SEQ ID NO:3 (Z-LEVD) 2 -Rhodamine 110, SEQ ID NO:9 (Z-IEPD) 2 -Rhodamine 110, SEQ ID NO:23 (Z-VEPD
  • Preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula VII and include, but are not limited to:
  • N-(Z-YVAD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:2
  • SEQ ID ⁇ O:9 N-(Z-DETD)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ O:4
  • SEQ ID NO: 8 N-(Z-IEPD)-.V -acetyl-Rhodamine 110.
  • N-(Z-SHVD)-N '-acetyl-Rhodamine 110 SEQ ID NO: 10
  • N-(Z-DGPD)-N '-acetyl-Rhodamine 110 SEQ ID NO: 12
  • SEQ ID NO: 13 N-(Z-DEPD)-N '-acetyl-Rhodamine 110, SEQ ID NO: 13
  • N-(Z-DSLD)-N'-acetyl-Rhodamine 110 SEQ ID NO: 17 N-(Z-DVPD)-N '-acetyl-Rhodamine 110, SEQ ID NO : 18 N-(Z-DEAD)-N '-acetyl-Rhodamine 110, SEQ ID NO: 19 N-(Z-DSYD)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ O:20 N-(Z-ELPD)-N'-acetyl-Rhodamine 110, SEQ ID ⁇ O:21 N-(Z-VEID)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ O:26 N-(Z-IETD)-N -acetyl-Rhodamine 110, SEQ ID ⁇ O:24
  • N-(Z-TD)-N '-acetyl-Rhodamine 110 N-(Z-AD)-N '-acetyl-Rhodamine 110
  • N-(Z-VAD)-N '-acetyl-Rhodamine 110 N-(Boc-WEHD)-N '-acetyl-Rhodamine 1 10, SEQ ID ⁇ O:l
  • SEQ ID ⁇ O:2 N-(Ac-LETD)-N '-acetyl-Rhodamine 1 10, SEQ ID ⁇ O:8 N-(Ac-LEHD)-N '-acetyl-Rhodamine 110.
  • SEQ ID ⁇ O:3 N-(Z-DEVD)-N'-methoxycarbonyl-Rhodamine 1
  • SEQ ID ⁇ O:2 N-(Ac-LETD)-N '-acetyl-Rhodamine 1 10
  • SEQ ID ⁇ O:8 N-(Ac-LEHD)-N '-acetyl-Rhodamine 110.
  • SEQ ID ⁇ O:3 N-(
  • N-(Z-LEVD)-N'-methoxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:9 N-(Z-LEHD)-N'-methoxycarbonyl-Rhodamine 1 10,
  • SEQ ID ⁇ O:3 N-(Ac-WEHD)-N'-methoxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O: l N-(Ac-YVAD)-N '-methoxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:2 N-(Ac-DEVD)-N '-methoxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-methoxycarbonyl-Rhodamine 1 10.
  • N-(Ac-DEHD)-N '-methoxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:6 N-(Ac-DETD)-N'-methoxycarbonyl-Rhodamine 1 10
  • SEQ ID ⁇ O:4 N-(Ac-LEVD)-N '-methoxycarbonyl-Rhodamine 110
  • SEQ ID ⁇ O:9 N-(Ac-LEHD)-N'-methoxycarbonyl-Rhodamine 1 10.
  • N-(Ac-VEHD)-N'-methoxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ N-(Ac-IEPD)-N '-methoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:23 N-(Z-WEHD)-N'-ethoxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O: l N-(Z-YVAD)-N'-ethoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:2 N-(Z-DEVD)-N'-ethoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-ethoxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-ethoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:l N-(Ac-YVAD)
  • N-(Ac-LEHD)-N'-octyloxycarbonyl-Rhodamine 1 10 SEQ ID ⁇ O:3 N-(Ac-LETD)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 8 N-(Ac-VEHD)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ N-(Ac-IEPD)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:23 N-(Z-WEHD)-V'-decyloxycarbonyl-Rhodamine 1 10. SEQ ID ⁇ O:l
  • SEQ ID ⁇ O:2 V-(Z-DEVD)-N'-decyloxycarbonyl-Rhodamine 110
  • SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-decyloxycarbonyl-Rhodamine 1 10
  • SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-decyloxycarbonyl-Rhodamine 1 10
  • SEQ ID ⁇ O:l N-(Ac-YVAD)-N'-decyloxycarbonyl-Rhodamine 1 10.
  • N-(Ac-DEVD)-N'-decyloxycarbonyl-Rhodamine 1 10 SEQ ID ⁇ O:5 N-(Ac-DEHD)-N ' -decyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:6 N-(Ac-DETD)-N'-decyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:4 N-(Ac-LEVD)-N'-decyloxycarbonyl-Rhodamine 1 10. SEQ ID ⁇ O:9 N-(Ac-LEHD)-N'-decyloxycarbonyl-Rhodamine 1 10. SEQ ID ⁇ O:3
  • SEQ ID NO: 8 N-(Ac-VEHD)-N ' -decyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ OJ N-(Ac-IEPD)-N'-decyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:23 N-(Z-WEHD)-N'-dodecyloxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:l N-(Z-YVAD)-N'-dodecyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:2
  • SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-dodecyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-dodecyloxycarbonyl-Rhodamine 1 10,
  • N-(Z-WEHD)-N'-(hexylthio)carbonyl-Rhodamine 1 10 SEQ ID ⁇ O: l N-(Z-YVAD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ O:2 N-(Z-DEVD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID NO: 1
  • SEQ ID NO: 8 N-(Ac-VEHD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ N-(Ac-IEPD)-N'-(hexylthio)carbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:2 N-(Z-DEVD)-N'-(octylthio)carbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:6 N-(Ac-DETD)-N'-(octylthio)carbonyl-Rhodamine 110
  • SEQ ID ⁇ O:2 N-(Z-LE(OAM)HD(OAM))-N '-acetyl-Rhodamine 1 10
  • SEQ ID ⁇ O:3 N-(Z-D(OAM)E(OAM)TD(OAM))-N'-acetyl-Rhodamine 1
  • SEQ ID ⁇ O:5 N-(Z-D(OMe)E(OMe)VD(OAM))-N '-acetyl-Rhodamine 1 10.
  • SEQ ID NO: 5 N-(Z-D(OMe)E(OMe)VD(OAM))-N '-acetyl-
  • N-(Z-D(OMe)E(OMe)VD)-N '-acetyl-Rhodamine 1 10.
  • SEQ ID NO: 5 N-(Z-VD(OAM))-N '-acetyl-Rhodamine 1 10, and N-(Z-E(OAM)VD(OAM))-N '-acetyl-Rhodamine 110.
  • Another preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula VIII and include, but are not limited to:
  • N-(Z-WEHDG)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ OJ1 N-(Z-YVADG)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ OJ2 N-(Z-LEHDG)-N '-acetyl-Rhodamine 1 10.
  • SEQ ID ⁇ OJ3 N-(Z-LEVDG)-N '-acetyl-Rhodamine 1 10.
  • SEQ ID NO: 74 N-(Z-DETDG)-N '-acetyl-Rhodamine 1 10, SEQ ID ⁇ OJ5
  • N-(Z-DEVDG)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Ac-LETDG)-N'-acetyl-Rhodamine 110, SEQ ID ⁇ OJ7 N-(Ac-LEHDG)-N'-acetyl-Rhodamine 110, SEQ ID ⁇ OJ3 N-(Ac-WEHDG)-N'-methoxycarbonyl-Rhodamine 110, SEQ ID ⁇ OJ1 N-(Ac-YVADG)-N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 72
  • N-(Ac-DEVDG)-N '-methoxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Ac-DEHDG)-N '-methoxycarbonyl-Rhodamine 1 10
  • SEQ ID ⁇ OJ8 N-(Z-WEHDGG)-N'-ethoxycarbonyl-Rhodamine 110
  • SEQ ID ⁇ OJ9 N-(Z-YVADG)-N'-ethoxycarbonyl-Rhodamine 1 10
  • SEQ ID ⁇ OJ2 N-(Z-DEVDG)-N'-ethoxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Z-LEVDG)-N'-ethoxycarbonyl-Rhodamine 110
  • SEQ ID ⁇ O.J4 N-(Ac-WEHDG)-N'-ethoxycarbonyl-Rhodamine 110
  • N-(Ac-WEHDG)-N'-hexyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ1 N-(Ac-YVADG)-N'-hexyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ2 N-(Ac-DEVDG)-N'-hexyloxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ OJ6 N-(Ac-DEHDG)-N'-hexyloxycarbonyl-Rhodamine 1 10
  • SEQ ID ⁇ OJ8 N-(Ac-WEHDG)-N'-octyloxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ OJ 1 SEQ ID ⁇ OJ 1
  • N-(Ac-DEVDG)-N'-(decylthio)carbonyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Ac-DEHDG)-N'-(decylthio)carbonyl-Rhodamine 110, SEQ ID ⁇ OJ8 N-(Ac-WEHDG)-N'-(dodecylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ1 N-(Ac-YVADG)-N'-(dodecylthio)carbonyl-Rhodamine 110, SEQ ID ⁇ OJ2 N-(Ac-DEVDG)-N '-(dodecylthio)carbonyl-Rhodamine 1 10, SEQ ID NO : 76
  • Another preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula IX and include, but are not limited to:
  • N-(GPG)-N'-octyloxycarbonyl-Rhodamine 110 N-(GP)-N'-ethoxycarbonyl-Rhodamine 110,
  • N-(MGG)-N'-octyloxycarbonyl-Rhodamine 110 N-(MGA)-N ' -octyloxycarbonyl-Rhodamine 1 10
  • N-(MA)-N '-ethoxycarbonyl-Rhodamine 110 N-G-N '-ethoxycarbonyl-Rhodamine 110,
  • N-(MG)-N'-(ethylthio)carbonyl-Rhodamine 110 N-G-N'-(ethylthio)carbonyl-Rhodamine 110,
  • N-(Ac-LM)-N '-ethoxycarbonyl-Rhodamine 110 N-(Boc-LM)-N'-hexyloxycarbonyl-Rhodamine 110, N-(Ac-LM)- ⁇ r -hexyloxycarbonyl-Rhodamine 1 10, N-(Boc-LM)-N'-(ethylthio)carbonyl-Rhodamine 110, N-(Ac-LM)-N'-(ethylthio)carbonyl-Rhodamine 110, N-(Ac-SL ⁇ FPIV)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:80 N-(Ac-SLNFPI)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:81 N-(Ac-SLNFP)-N'-octyloxycarbonyl-Rho
  • N-(Ac-IRGGG)-N '-octyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:97 N-(Ac-LVGGG)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:98 N-(Ac-MVGGG)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:99 N-(Ac-IVGGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 100 N-(Ac-LRGGG)-N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 101
  • N-(Ac-LRGGA)-N '-octyloxycarbonyl-Rhodamine 1 10.
  • SEQ ID NO: 102 N-(Ac-LRGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:55 N-(Z-LRGGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:101 N-(Z-LRGGA)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 102 N-(Z-LRGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:55
  • N-(Ac-LRGGG)-N'-ethoxycarbonyl-Rhodamine 1 10.
  • SEQ ID NO: 101 N-(Ac-LRGGA)-N'-ethoxycarbonyl-Rhodam ⁇ ne 1
  • SEQ ID NO: 102 N-(Ac-LRGG)-N'-ethoxycarbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:55 N-(Ac-LRGGG)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
  • SEQ ID NO: 101 N-(Ac-LRGGA)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
  • SEQ ID NO: 102 N-(Ac-LRGGA)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
  • N-(Ac-LRGG)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
  • SEQ ID ⁇ O:55 N-(Ac-LVLASSS)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 103 N-(Ac-LVLASS)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 104 N-(Ac-LVLAS)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 105 N-(Ac-LVLA)-N -octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:62
  • SEQ ID NO:l 10 N-(Z-Tbg-Tbg-NASS)-A'-octyloxycarbonyl-Rhodamine 110
  • SEQ ID NO: 108 N-(Z-Tbg-Tbg-NAS)-A '-octyloxycarbonyl-Rhodamine 1
  • SEQ ID ⁇ O:110 N-(Ac-Tbg-Tbg-NASS)-N'-ethoxycarbonyl-Rhodamine 110
  • SEQ ID NO: 109 N-(Ac-Tbg-Tbg-NA)-A '-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 1 10 N-(Ac-Tbg-Tbg-NASS)-N'-(ethylthio)carbonyl-Rhodamine 1 10
  • SEQ ID NO: 109 N-(Ac-Tbg-Tbg-NA)-A'-(ethylthio)carbonyl-Rhodamine 110 SEQ ID NO: 110
  • SEQ ID ⁇ O:l 1 1 N-(Ac-DIVPCSMST)-V'-octyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:l 12 N-(Ac-IVPCSMST)-N ' -octyloxycarbonyl-Rhodamine 110
  • SEQ ID ⁇ O:114 N-(Ac-IVPCSM)-N '-octyloxycarbonyl-Rhodamine 110 SEQ ID NO: 115
  • N-(Ac-IVPCS)-N '-octyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:l 16 N-(Ac-IVPC)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:69 N-(Z-IVPCSMST)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:l 13 N-(Z-IVPCSMS)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:l 14 N-(Z-IVPCSM)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 115 N-(Z-IVPCS)-N -octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:l 16
  • N-(Ac-IVPCS)-N'-ethoxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:l 16 N-(Ac-IVPCSMS)-N'-(ethylthio)carbonyl-Rhodamine 110, SEQ ID NO: 114
  • Z is benzyloxycarbonyl
  • BOC is tert.-butoxycarbonyl
  • Ac is acetyl
  • Tbg is t-butylglycine
  • AM is acetoxymethyl.
  • Preferred novel fluorescent dyes of the present invention are compounds having Formula VI and include, but are not limited to:
  • N-hexanoyl-Rhodamine 110 N-octanoyl-Rhodamine 110,
  • N-ethoxycarbonyl-Rhodamine 110 N-butoxycarbonyl-Rhodamine 110,
  • N-dodecyloxycarbonyl-Rhodamine 110 N-benzyloxycarbonyl-Rhodamine 110,
  • N-decanesulfonyl-Rhodamine 110 N-dodecanesulfonyl-Rhodamine 110,
  • N-trifluoromethanesulfonyl-Rhodamine 116 N-octanesulfonyl-Rhodamine 116,
  • Typical aryl groups are C 6 . I0 aryl groups including phenyl, naphthyl, fluorenyl and the like, any of which may be substituted with halo or alkyl groups.
  • Typical alkyl groups are C,_ 10 alkyl groups including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and branced chain isomers thereof.
  • Typical acyl (alkanoyl) groups are C 2 .. 0 alkanoyl groups such as acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and the like as well as the branched chain isomers thereof.
  • Typical biologically acceptable salts of the compounds of the invention include the sodium, potassium, ammonium, TRIS and the like.
  • Certain of the compounds of the present invention may be in tautomeric forms, particularly in the y-portion of Formula I.
  • the invention includes all such tautomers.
  • the invention also includes stereoisomers, the racemic mixtures of such stereoisomers as well as the individual entantiomers that may be separated according to methods that are well known to those of ordinary skill in the art.
  • the compounds of this invention may be prepared using methods known to those skilled in the art. Specifically, compounds with Formulae I-III can be prepared as illustrated by exemplary reactions in Schemes 1 -5.
  • Scheme 1 is the least preferred method since deprotection with HBr/HOAc led to the removal of both the t-butoxy and benzyloxycarbonyl (Z) groups, which makes the next coupling reaction complicated. Thus, where the t-butoxy group is desired, it must be reintroduced.
  • N-(9-fluorenylmethoxycarbonyl) (fmoc) group is employed as the N-blocking group (Scheme 2), it can be selectively removed with morpholine, piperidine or other amine base without removing the t-butoxy protecting groups, thus allowing for the ready introduction of additional Z-blocked amino acids or peptides (see Schemes 2-4).
  • the final Z- blocked compounds can be selectively deprotected with trifluoroacetic acid (TFA) to remove the t-butoxy group without removing the Z group.
  • TFA trifluoroacetic acid
  • the invention also relates to a method for the preparation of a compound of Formula III, comprising
  • -(AA) ⁇ is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET. SHV, DEL, DGP, DEP, DGT, DL ⁇ , DEE, DSL, DVP, DEA, DSY, ELP, VED, IEP or IET.
  • the amino acid is substituted by a carboxy group, it is protected with a OBu-t protecting group which is removed in the final step.
  • the condensation reaction may be carried out using any conventional condensing agent that is used for peptide synthesis.
  • the condensing agent is l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), or 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ).
  • the solvent for the reaction may be pyridine or dimethylformamide (DMF).
  • the reaction is generally carried out at room temperature.
  • the Fmoc group is generally removed by treatment with morpholine, piperidine or other amine base, in a polar aprotic solvent such as DMF.
  • a polar aprotic solvent such as DMF.
  • the morpholine is added in excess, and the reaction is carried out at room temperature.
  • ⁇ , ⁇ -Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz) is another N- blocking group which can be used in the place of fmoc.
  • Ddz-L-aspartic acid ⁇ -t-butyl ester can be used in place of N-fmoc-L-aspartic acid ⁇ -t-butyl ester.
  • Ddz can be cleaved selectively in the presence of t-butoxy group by 1 %TFA in methylene chloride.
  • the OBu-t group is removed with trifluoroacetic acid in an aprotic solvent such as methylene chloride at room temperature.
  • the invention also relates to a method for the preparation of a compound of Formula VII, comprising
  • -(AA) repeat is WEH.
  • Compounds of Formula VII also can be prepared using an acyl (alkanoyl) chloride in place of acetic anhydride, such as acetyl chloride, hexanoyl chloride, octanoyl chloride and decanoyl chloride.
  • acyl alkanoyl
  • reagents can be used in place of acetic anhydride include, but are not limited to carbamyl chloride such as dimethylcarbamyl chloride, diethylcarbamyl chloride and N-methyl-N- hexylcarbamyl chloride; chloroformate such as methyl chloroformate, ethyl chloroformate, octyl chloroformate, 2-butoxyethyl chloroformate and 2,5,8- trioxadecyl chloroformate; chlorothiolformate such as methyl chlorothiolformate, ethyl chlorothiolformate, octyl chlorothiolformate; alkyl, haloalkyl and aralkyl sulfonyl halides such as methanesulfonyl chloride, octanesulfonyl chloride, trifluoromethanesulfonyl chloride and tosylchloride.
  • the reaction is carried out in the presence of a base, such as (Et) 3 ⁇ , (i-Pr) 2 -NEt or pyridine.
  • a base such as (Et) 3 ⁇ , (i-Pr) 2 -NEt or pyridine.
  • the preferred solvent is DMF.
  • the reaction is generally carried out at room temperature.
  • the ratio of anhydride or acyl chloride to Rhodamine is about 1 :1.
  • the condensation reaction may be carried out using any conventional condensing agent that is used for peptide synthesis.
  • the condensing agent is EDC or EEDQ
  • the solvent for the reaction is pyridine or dimethylformamide (DMF).
  • the reaction is generally carried out at room temperature.
  • the ratio of condensing agent to N-acetyl-Rhodamine is about 3:1 and the ratio of protected amino acid or peptide to N-acetyl-Rhodamine or N- (Asp(OBu-t))-N'-acetyl-Rhod.amine is about 3:1.
  • compounds of Formula VI provide fluorescent dyes which can be condensed with any peptide or other structure for the preparation of fluorogenic or fluorescent compounds which are substrates for proteases or peptidases.
  • compounds of Formula VII also can be prepared by first condensing a peptide with a Rhodamine to give N-peptide-Rhodamine, then reacting the N-peptide-Rhodamine with acetyl anhydride or other acylating reagent to give for example, N-acetyl-N'-peptide-Rhodamine.
  • a) peptides in general are much more expensive than acyl chlorides or anhydrides
  • the condensation reaction between peptide and Rhodamine is not an efficient reaction. For these reasons it is preferred to attach the peptide to N-acetyl- Rhodamine rather than attach the acyl group to N-peptide-Rhodamine.
  • the invention relates to a method for determining whether a test substance has an effect on an enzyme involved in the apoptosis cascade in a test cell, comprising
  • test substance either interacts with an external membrane receptor of the cell or is taken into the cell and the reporter compound is taken into the cell
  • Activators either by direct or indirect mechanisms, of enzymes involved in the apoptosis cascade include but are not limited to known chemotherapeutic agents, such as etoposide (Yoon HJ, Choi IY, Kang MR, Kim SS, Muller MT, Spitzner JR, Chung IK (1998), Biochim Biophys Ada 1395:110-120) and doxorubicin (Gamen S, Anel A, Lasierra P, Alava MA, Martinez-Lorenzo MJ, Pineiro A, Naval J (1997), FEBS
  • chemotherapeutic agents such as etoposide (Yoon HJ, Choi IY, Kang MR, Kim SS, Muller MT, Spitzner JR, Chung IK (1998), Biochim Biophys Ada 1395:110-120) and doxorubicin (Gamen S, Anel A, Lasierra P, Alava MA, Martinez-Lorenzo MJ, Pineiro A, Naval J
  • Inactivators either by direct or indirect mechanisms, of enzymes involved in the apoptosis cascade include but are not limited to endogenous proteins including Bcl-2 (Joensuu H, Pylkkanen L, Toikkanen S (1994;, Am. J. Pathol 5: 1 191- 1198), the viral produced agent p35 (Miller LK (1997;, J. Cell Physiol 173: 178-
  • the invention relates to the use of the reporter compounds having Formulae I-III, V, VII and VIII in whole-cell assays, using whole cells or tissue samples which have been induced to undergo apoptosis, to screen for compounds that inhibit either directly or indirectly an enzyme or enzymes involved in apoptosis (programmed cell death).
  • These screening assays using compounds having Formulae I-III, V, VII and VIII are expected to lead to discovery of new drugs or new uses for known drugs that slow or block cell death in a variety of clinical conditions in which the loss of cells, tissues or entire organs occurs.
  • the reporter compounds having Formulae I-III, V, VII and VIII and the screening assays of the present invention can be used to identify drugs that reduce or prevent cell death in the nervous system (brain, spinal cord, and peripheral nervous system) under various conditions of ischemia and excitotoxicity, including, but not limited to, focal ischemia due to stroke and global ischemia due to cardiac arrest.
  • the screening assays can also be used to identify compounds that reduce or prevent cell death in the nervous system due to traumatic injury (such as head trauma or spinal cord injury), viral infection or radiation-induced nerve cell death (for example, as a side-effect of cancer radiotherapy) or environmental toxicity (e.g. by certain halogenated hydrocarbon).
  • the screening assays can also be used to identify cell death inhibitors which are useful to reduce or prevent cell death in a range of neurodegenerative disorders, including but not limited to Alzheimer's disease, Huntington's Disease, Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis, and spinobulbar atrophy.
  • the screening assays of this invention can be used to identify compounds that prevent cell death in any condition which potentially results in the death of cardiac muscle. This includes myocardial infarction, congestive heart failure and cardiomyopathy.
  • One particular application of the screening assay is to identify compounds which reduce or prevent myocardial cell death that occurs in certain viral infections of the heart.
  • the screening assays of the invention can be used to identify compounds which prevent cell death of retinal neurons that occurs in disorders associated with increased intraocular pressure (such as glaucoma) or retinal disorders associated with the aging process (such as age-related macular degeneration).
  • the assays can also be used to identify compounds which treat hereditary degenerative disorders of the retina, such as retinitis pigmentosa.
  • the screening assays of the invention can also be used to identify cell death inhibitors that can be used to reduce or prevent premature death of cells in the immune system, and are particularly useful in identifying inhibitors which are useful in treating immune deficiency disorders, such as acquired immune deficiency syndrome (AIDS), severe combined immune deficiency syndrome (SCIDS) and related diseases.
  • the screening assays can also be used to identify cell death inhibitors that can be used to treat radiation-induced immune suppression.
  • the screening assays of the invention can also be used to identify drugs useful in organ transplantation procedures. Transplantation of human organs and tissues is a common treatment for organ failure. However, during the transplantation process, the donor organ or tissue is at risk for cell death since it is deprived of its normal blood supply prior to being implanted in the host. This ischemic state can be treated with cell death inhibitors by infusion into the donor organ or tissue, or by direct addition of the cell death inhibitors to the organ/tissue storage medium. Such cell death inhibitors can be identified using the screening assays described in this invention. Cell death inhibitors may also be used to reduce or prevent cell death in the donor organ/tissue after it has been transplanted to protect it from the effects of host immune cells which kill their targets by triggering apoptosis.
  • the screening assays described in this invention can be used to identify cell death inhibitors useful in protecting transplanted organs from rejection.
  • the cytoprotective effects of cell death inhibitors can also be used to prevent the death of human or animal sperm and eggs used in in vitro fertilization procedures. These inhibitors can be used during the harvesting process and can also be included in the storage medium.
  • Cell death inhibitors useful for application in fertilization procedures can be identified using the screening assay methods described in this invention.
  • Mammalian cell lines and yeast cells are commonly used to produce large amounts of recombinant proteins (such as antibodies, enzymes or hormones) for industrial or medicinal use.
  • the lifespan of some of these cell lines is limited due to growth conditions, the nature of the recombinant molecule being expressed (some are toxic) and other unknown factors.
  • the lifespans of industrial cell lines can be extended by including cell death inhibitors in the growth medium.
  • Cell death inhibitors useful in extending the life span of cell lines can be identified using the screening assay procedures described in this invention.
  • hair follicle regression (referred to as catagen) may be due at least partially to apoptosis. Therefore, it is possible that cell death inhibitors can be used to treat hair loss that occurs due to various conditions, including but not limited to male-pattern baldness, radiation-induced or chemotherapy-induced hair loss, and hair loss due to emotional stress. There is also evidence that apoptosis may play a role in the loss of hair color. Therefore, it is possible that cell death inhibitors can also be used in treating cases of premature graying of the hair. Cell death inhibitors useful in treating or preventing hair loss or graying of the hair can be identified using the screening assay procedures described in this invention.
  • the death of skin epithelial cells can occur after exposure to high levels of radiation, heat or chemicals. It is possible that cell death inhibitors can be used to reduce or prevent this type of skin damage. In one particular application, cell death inhibitors can be applied in an ointment to treat acute over-exposure to the sun and to prevent blistering and peeling of the skin. Cell death inhibitors useful in treating or preventing death of skin cells can be identified using the screening assay procedures described in this invention.
  • reporter compounds having Formulae I-III, V, VII and VIII in whole-cell assays using live or dead whole cells or tissue samples to screen for compounds that stimulate, either directly or indirectly, an enzyme or enzymes involved in apoptosis.
  • these screening assays using compounds having Formulae I-III, V, VII and VIII are expected to lead to discovery of new drugs or new uses for known drugs that act as anti-cancer agents in diseases such as cancers, tumors and cell hyperplasias etc.
  • Compounds that may be found using the screening assays and reagents described herein are useful for treatment of cancers, tumors or tissue hyperplasias including but not limited to cancers or tumors of the brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands, joints, bones and skin.
  • reporter compounds having Formulae I-III, V, VII and VIII in whole-cell assays using yeast and other fungi, and bacteria to screen compound libraries for anti-fungal or anti-bacterial drugs that act by inducing, either directly or indirectly, the caspase cascade or other enzymes involved in apoptosis in those cells.
  • Another important aspect of the invention is to use the reporter compounds having Formulae I-III, V, VII and VIII to monitor the therapeutic effects of therapeutic agents or treatments given to patients with the aim of reducing, preventing or treating maladies in which apoptotic cell death is either a cause or a result.
  • Another important aspect of the present invention is to use the reporter compounds having Formulae IX to screen for HIV protease inhibitors in HIV infected cells, comprising
  • test substance either interacts with an external membrane receptor or is taken into said cell and the reporter compound is taken into the cell
  • Yet another important aspect of the present invention is to use the reporter compounds having Formulae IX to diagnose HIV infection, comprising (a) contacting a test cell from an individual suspected of having HIV infection with the reporter compound according to the invention under conditions whereby the reporter compound is taken into the cell, and
  • the reporter compounds having Formula IX of the present invention can be used to screen for adenovirus protease inhibitors in adenovirus infected cells.
  • the reporter compounds having Formula IX of the present invention also can be used to screen for herpes simplex virus type-1 (HSV-1) protease inhibitors in HSV-1 infected cells.
  • HSV-1 herpes simplex virus type-1
  • the reporter compounds also can be used to screen for human cytomegalovirus (HCMV) protease inhibitors in HCMV infected cells; to screen for hepatitis C virus (HCV) protease inhibitors in HCV infected cells; to screen for DPP-IV inhibitors in T-cells; as well as to screen for type-2 methionine aminopeptidase (MetAP-2) inhibitors in endothelial cells.
  • HCMV human cytomegalovirus
  • HCV hepatitis C virus
  • MethodAP-2 type-2 methionine aminopeptidase
  • the reporter compounds having Formula IX of the present invention also can be used to diagnose adenovirus, herpes simplex virus type-1, human cytomegalovirus and hepatitis C virus.
  • compositions within the scope of this invention include all compositions wherein the fluorogenic or fluorescent compounds of the present invention are contained in an amount which are effective to achieve its intended purpose.
  • the fluorogenic or fluorescent substrate compounds may be applied to cells or cell lines from mammals, e.g. humans, or other animals by incubating the cells or tissues containing the cells with the fluorogenic or fluorescent substrate at a concentration of about 0.01 nanomolar to about 1 molar, or an equivalent amount of a salt or proreporter molecule thereof in a physiologically compatible buffer.
  • buffers include cellular growth medias, an example for leukemia derived cancer cells being RPMI-1640 with or without 10% fetal bovine serum.
  • Other known cellular incubation buffers could involve isotonic solutions buffered with either phosphate or HEPES.
  • the cells can be derived from any organ or organ system for which it is desirable to find— by- means of the screening assays— drugs that could be useful in treating apoptosis- mediated disorders, e.g., neuronal cell death, heart disease, liver disease, retinal disorders, kidney, joint and bone diseases, immune system disorders, cancers, tumors and tissue hyperplasias etc.
  • apoptosis- mediated disorders e.g., neuronal cell death, heart disease, liver disease, retinal disorders, kidney, joint and bone diseases, immune system disorders, cancers, tumors and tissue hyperplasias etc.
  • Suitable solubilizers may be used for presenting the fluorogenic or fluorescent compounds of the present invention to tissues, cells or cell lines.
  • Suitable solubilizers include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts and alkaline solutions.
  • suspensions of the compounds as appropriate oily suspensions may be presented to the cells or tissues.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400) or dimethylsulfoxide (DMSO) or another suitable solubilizer.
  • the suspension or solution may also contain stabilizers.
  • electroporation or presentation of the reporter molecules in liposomes or detergents may be used to enhance the cell permeability of the fluorogenic or fluorescent reporter molecules.
  • the cells are contacted with the reporter compounds of the invention and the test substance for about 30 minutes to about 5 hours, most preferably, about 1 hour.
  • the invention also relates to the pro-reporter derivatives of the compounds of the invention.
  • pro-reporter derivatives include compounds which are cleaved in situ by endogenous enzymes to give the compounds of Formulae I-III, V, and VII-IX.
  • Such pro-reporter derivatives include lower alkyl esters of carboxyl-containing amino acid residues such as Asp and Glu.
  • Especially preferred pro-reporter derivatives include the methyl esters and acetoxymethyl (AM) esters of Asp- and Glu-containing compounds.
  • the following examples demonstrate usefulness of the invention in measuring the activity of caspases and other enzymes involved in apoptosis in cells and tissues.
  • Rhodamine 110 500 mg, 1.36 mmol
  • DMF dimethyl methoxysulfoxide
  • acetic anhydride 167 mg. 1.64 mmol was added dropwise to the above solution.
  • the reaction solution was stirred at room temperature for 24 h. and it was then diluted with 100 mL of water and extracted with ethyl acetate (3 x 50 mL).
  • Rhodamme 1 10 (3.00 g, 8.18 mmol) dissolved in dimethylformamide (140 mL) at -50 °C was added N,N-diisopropylethylamine (1.27 g, 1.2 mmol), then ethyl chloroformate (1.07 g, 9.81 mmol) was added dropwise to the above solution.
  • the reaction solution was then slowly warmed to room temperature and kept stirring for 5 h. It was then diluted with 200 mL of ice water and extracted with ethyl acetate (3 x 50 mL).
  • Rhodamine 110 SEQ ID ⁇ O:5
  • Rhodamine 110 SEQ ID ⁇ O:5
  • Rhodamine 1 16 (458.8 mg, 1.0 mmol), NN-diisopropylethylamine (129.3 mg, 1.0 mmol) and acetic anhydride (122 mg, 1.2 mmol) (as described in
  • Example 25 was obtained 141 mg (9.4%>) of the title compound as colorless solid.
  • ⁇ ⁇ MR (CDC1 3 ): ⁇ 8.01 (d, J 7.5 Hz, IH), 7.69-7.62 (m, 3H), 7.24-6.36 (m. 7H), 3.95 (bs, 2H), 3.28 (s, 3H), 2.87 (s, 3H), 1.95 (bs, 3H).
  • Rhodamine 110 500 mg, 1.36 mmol
  • dimethylformamide (12 mL) at -61 °C NN-diisopropylethylamine (264 mg, 2.04 mmol)
  • ethyl chlorothiolformate 204 mg, 1.64 mmol
  • the reaction solution was then slowly warmed to room temperature and kept stirring for 1 h. It was then diluted with 100 mL of ice water and extracted with ethyl acetate (3 x 30 mL).
  • N-ethoxycarbonyl Rhodamine 110 (58 mg, 0.135 mmol) (according to Example 1) was obtained the title compound (70 mg, 83%) as a solid.
  • Rhodamine 1 10 0.5 g, 1.36 mmol
  • DMF 15 ml
  • NN-diisopropylethylamine 0.25 ml
  • N-hexyl-N- methylcarbamyl chloride in DMF (2.05 mmol.
  • the reaction mixture was stirred at -61°C for 1 h, then was allowed to warm up to rt.
  • the reaction mixture was further stirred at rt for 14 h, and then was partitioned between aqueous saturated ⁇ H 4 C1 solution and ethyl acetate (2 x 50 ml).
  • Rhodamine 110 and N-acetyl-Rhodamine 110 were measured in a fluorometric assay.
  • the fluorescent signal is read in a spectrofluorometer or in a fluorometric microtiter plate reader at excitation wavelength of 485 and emission 530. Using this assay, the relative fluorescent values were determined for the two fluorescent moieties.
  • Rhodamine 1 10
  • N-acetyl-Rhodamine 110 is a stable and efficient fluorescent indicator.
  • Modified Rhodamine dyes were evaluated using both conventional spectrometry and spectrofluorometry. For both types of analysis the dyes were dissolved in either methanol or 50 mM Tris at final dye concentrations ranging from 10 nM to 100 ⁇ M. An absorbance spectrum from wavelengths of 200 ran to 700 nm was determined for each dye using a Beckman DU-7000 spectrophotometer. The dyes all had absorbance peaks at around 470 to 480 nm. This wavelength was chosen as the fluorescence excitation wavelength and a full fluorescence emission spectrum was determined using a Hitachi F-2000 spectrofluorometer. For each dye, the emission peak was around 520 nm and the fluorescent output was measured under the conditions tested (see Table 1).
  • HL-60 cells were placed in 5 ml of Iscove's medium (without serum or phenol-red) containing 10 ⁇ M or 50 ⁇ M N-octyloxycarbonyl-Rhodamine 110, N- decyloxycarbonyl-Rhodamine-110, N-dodecyloxycarbonyl-Rhodamine-110, N- hexyloxycarbonyl-Rhodamine-110, N-(ethylthio)carbonyl-Rhodamine 110 or Rhodamine 110.
  • the cells were incubated for varying times at 37°C in a C0 2 incubator, recovered by centrifugation, and washed in 50 mL of ice-cold medium.
  • N-octyloxycarbonyl-Rhodamine 1 10 (Fig. 1A), N- decyloxycarbonyl-Rhodamine 1 10 (Fig. IB), and N-dodecyloxycarbonyl- Rhodamine 110 (Fig. 1C) stained HL-60 cells intensely and there was almost no leakage of the dye into the medium.
  • N-Hexyloxycarbonyl-Rhodamine 1 10 (Fig.
  • Rhodamine 1 10 (Fig. IF) stained cells rapidly, but the dye quickly leaked out of the cells, resulting in a low intensity of cellular staining and a high degree of fluorescence in the medium containing the cells. Therefore, the modified Rhodamine dyes are superior to Rhodamine 110 since they are readily taken up by HL-60 cells and are retained within the cells for at least 30 minutes.
  • N-(Z-VD)-N '-acetyl-Rhodamine 110 The activities of N-(Z-VD)-N '-acetyl-Rhodamine 110, N-(Z-VAD)-N'- acetyl-Rhodamine 110, N-(Z-DEVD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:5,
  • N-(Z-YVAD)-N-acetyl-Rhodamine 110 SEQ ID ⁇ O:2, (Z-VAD) 2 -Rhodamine 110 and (Z-YVAD) 2 -Rhodamine 110 SEQ ID NO:2 as synthetic substrates for recombinant CPP32 and ICE were measured in a fluorometric enzyme assay.
  • Recombinant CPP32 protein and ICE protein were prepared by expressing DNA clones encoding these enzymes in an insect host cell (sf9 cells) using baculovirus as the vector. See. Webb, N.R. et al. "Expression of proteins using recombinant Baculovirus," Techniques 2:173-188 (1990).
  • CPP32 and ICE dependent substrate cleavage was measured using the following buffer conditions: 100 mM HEPES pH 7.5, with 10% sucrose, 1% CHAPS, 5 mM glutathione, and 1-100 ⁇ M test substrate.
  • Nonspecific enzyme cleavage was determined with the use of the specific CPP32 and ICE inhibitors consisted of an oligomer with the sequence Asp-Glu- Val-Asp or Tyr-Val-Ala-
  • the assay for enzyme activity was typically carried out at 37°C for 60 minutes.
  • Table 4 lists the K_- and V max values for N-(Z-VD)-N '-acetyl-Rhodamine 1 10, N-(Z-VAD)-N '-acetyl-Rhodamine 110, N-(Z-DEVD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:5, N-(Z-YVAD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:2, (Z-
  • N-(Z-VD)-N'-Ac-Rhodamine 110 60 1 1 NA N-(Z-VAD)-N'-Ac-Rhodamine 1 10 NA 70 4 N-(Z-DEVD)-N'-Ac-Rhodamine 1 10 154 160 12 9 SEQ ID ⁇ O:5
  • SEQ ID NO:2 NA no activity observed at 1- 100 ⁇ M substrate, 37°C, 3 h incubation
  • N-(Z-DEVD)-N '-acetyl- Rhodamine 110 SEQ ID ⁇ O:5 is an efficient substrate for both ICE and CPP32. Also shown is that N-(Z-VD)-N '-acetyl-Rhodamine 1 10 is an efficient substrate for CPP32 and not for ICE and that N-(Z-VAD)-N '-acetyl-Rhodamine 1 10, N-(Z- YVAD)-N '-acetyl-Rhodamine 1 10 SEQ ID ⁇ O:2, (Z-VAD) 2 -Rhodamine 1 10 and (Z-YVAD) 2 -Rhodamine 110 SEQ ID NO:2 are efficient substrates for ICE and not for CPP32.
  • the caspase substrates were assayed by recombinant caspase-3 and by lysates prepared from apoptotic HL-60 cells.
  • the assays were carried out at 37°C in 96-well plates in a 100 ⁇ L incubation containing 30 ⁇ L of caspase-3 preparation or cell lysate, 10 ⁇ M or 50 ⁇ M of the substrate, and caspase assay buffer (40 mM 1 ,4-piperazinebis(ethansulfonic acid) (PIPES, Aldrich Chemical Company) pH 7.2; 100 mM NaCl; 10% sucrose; 0.1% CHAPS; 1 mM EDTA; 10 mM DTT).
  • caspase assay buffer 40 mM 1 ,4-piperazinebis(ethansulfonic acid)
  • N-Ac-DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5 was tested using apoptotic HL-60 and Jurkat cells. These whole-cell assays were carried out in two stages: 1) induction of apoptosis; 2) incubation with the substrate. For HL-60 cells, apoptosis was induced by treatment with 10 ⁇ g/ml vinblastine for 4 hours. Control samples were treated with DMSO. For Jurkat cells, apoptosis was induced by treatment with 500 ng/ml agonistic antiFas antibody for 2 hours. Control samples were treated with PBS.
  • the cells were incubated with 50 ⁇ M N-Ac-DEVD-N-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5 in caspase assay buffer (40 mM PIPES, pH 7.4; 100 mM NaCl; 10% sucrose; 1 mM EDTA; 10 mM DTT).
  • caspase assay buffer 40 mM PIPES, pH 7.4; 100 mM NaCl; 10% sucrose; 1 mM EDTA; 10 mM DTT.
  • the cells were than transferred to a glass microslide and viewed by epifluorescent illumination on a Nikon inverted microscope.
  • vinblastine-treated HL-60 cells were intensely stained by N-Ac-DEVD-N'- octyloxycarbonyl R110 SEQ ID ⁇ O:5.
  • DMSO-treated cells also showed some staining (Fig. 3B). Although the intensity of the signal was significantly less than that of vinblastine-treated cells.
  • HL-60 cells treated with 50 ⁇ M Ac-DEVD-CHO SEQ ID NO: 5 during the assay stage (Fig. 3C) showed almost no fluorescent signal, indicating that the staining observed in vinblastine-treated cells is almost entirely due to caspase-mediated cleavage.
  • Jurkat cells induced to undergo apoptosis by antiFas (Fig. 3D) also showed intense staining by N-Ac-DEVD-N'- octyloxycarbonyl R1 10 SEQ ID ⁇ O:5, while control cells showed only light staining (Fig. 3F).
  • N-Ac-DEVD-N'- octyloxycarbonyl Rl 10 SEQ ID ⁇ O:5 can be used to measure apoptosis in intact cells and that the signal obtained from N-Ac-DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID NO: 5 is caspase-dependent.
  • N-Ac-LEVD-N-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9 was assayed by recombinant human caspase-3, 6, 7, and 8.
  • the assays were carried out at 37°C in 96-well plates in a 100 ⁇ L incubation containing recombinant human caspase, 10 ⁇ M of N-Ac-LEVD-N'-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9, and caspase assay buffer (40 mM PIPES, pH 7.2; 100 mM NaCl; 10% sucrose; 0.1% CHAPS; 1 mM EDTA; 10 mM DTT).
  • caspase-6 and caspase-8 cleave N-Ac-LEVD-N'-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9 to give an easily measured fluorescent signal (signal to background ratios of about 13 for caspase-6 to about 26 for caspase-8).
  • Caspase-3 cleaved N-Ac-LEVD-N'-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9 less efficiently, yielding a signal that was about 5-fold above the enzyme blank value.
  • Caspase-7 gave virtually no signal.
  • Aminopeptidases are present in many cells and sequentially remove unblocked amino acid residues from peptides, starting from the N-terminus. Peptides with blocked amino termini are not cleaved.
  • HL-60 lysates were prepared by homogenizing HL-60 cells in caspase buffer, and the ability of these lysates to cleave N-Z-G-N'-octyloxycarbonyl Rl 10 and N-G-N'-octyloxycarbonyl- Rl lO was tested in a microtiter plate assay.
  • Figure 6 shows that HL-60 cell lysates readily cleaved N-G-N'-octyloxycarbonyl Rl l O, and the size of the signal was dependent on the concentration of substrate. By contrast, no signal was generated by HL-60 cell lysates from N-Z-G-N'-octyloxycarbonyl-Rl 10.
  • Drugs that stimulate the caspase cascade in the absence of Fas ligand may be useful, for example, as anti-cancer chemotherapeutic agents.
  • the assay described in Example 78 may be used to screen for drugs that stimulate the caspase cascade by carrying out the assay under similar conditions as in Example 78, except that a known or unknown compound with known or unknown anti- cancer or anti-tumor activity replaces the Fas ligand reagent.
  • fluorescence assay in screening for drugs that inhibit or potentiate the caspase cascade stimulated with Fas ligand or another apoptosis inducer.
  • Drugs that inhibit the caspase cascade may be useful in treating degenerative and other diseases caused by or associated with an inadequate activation of the caspase cascade.
  • Drugs that potentiate the action of another caspase stimulator such as e.g. Fas ligand or an anti-cancer drug or agent, may be suitable to treat cancers or tumors caused by or associated with an inappropriate function of the caspase cascade.
  • the assays and reagents described in this invention may be used to screen for drugs that either inhibit or potentiate the caspase cascade in cells by performing the assay as described in Example 78 using Fas ligand or any other agent that stimulates the caspase cascade or other apoptosis pathway in the presence of a test substance that inhibits or potentiates or acts synergistically with the action of the first apoptosis or caspase cascade inducer.
  • the fluorescence assays described in this invention permit chemosensitivity or drug resistance testing of cancer or tumor cells or tissue samples taken from individual cancer or tumor patients.
  • a fluorescence assay using a cancer cell or tissue sample taken from a patient may be conducted as described Example 78.
  • different drugs with known or unknown chemotherapeutic activity can be tested for their capacity to stimulate the caspase cascade.
  • the results from this assay provide information that can be used to design an optimal chemotherapeutic drug treatment regimen for the patient.
  • HL-60 cells were placed in 5 ml of Iscove's medium (without serum or phenol-red) containing 10 ⁇ M N-G-N -octyloxycarbonyl-Rl 10 or 10 ⁇ M N-Z-G— N-octyloxycarbonyl-Rl 10.
  • Iscove's medium without serum or phenol-red
  • Three million HL-60 cells were incubated for 3 hours at 37 °C in a C0 2 incubator, recovered by centrifugation, and washed in 50 ⁇ L of ice-cold medium. The cells were re-centrifuged and the final pellet was resuspended in 50 ⁇ L of fresh medium.

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Abstract

La présente invention concerne de nouveaux colorants fluorescents, de nouvelles molécules fluorogènes et fluorescentes et de nouveaux procédés de dosages enzymatiques qui peuvent être utilisés pour détecter l'activité des caspases et d'autres enzymes impliqués dans l'apoptose des cellules entières, des lignées cellulaires et des échantillons tissulaires dérivés d'un organisme vivant ou d'un organe. Les molécules reporters et les procédés de dosage peuvent être utilisés dans des procédures de dosage de médicaments pour identifier des composés qui jouent le rôle d'inhibiteurs ou d'inducteurs des caspases en cascase dans les cellules entières ou les tissus. Les réactifs et les dosages décrits ici permettent de déterminer la chimiosensibilité de cellules cancéreuses de l'homme au traitement par des médicaments chimiothérapeutiques. La présente invention a également pour objet de nouvelles molécules reporters fluorogènes et fluorescentes et de nouveaux procédés de dosage enzymatiques qui peuvent être utilisés pour détecter l'activité de la méthionine aminopeptidase de type 2, du dipeptidyle peptidase IV, du calpain, de l'aminopeptidase, la protéase du VIH, la protéase d'adénovirus, la protéase HSV-A, la protéase HCMV et la protéase HCV.
PCT/US1998/021231 1997-10-10 1998-10-09 Nouvelles molecules reporters fluorescentes, applications de ces molecules et dosages des caspases WO1999018856A1 (fr)

Priority Applications (12)

Application Number Priority Date Filing Date Title
AU10722/99A AU754634B2 (en) 1997-10-10 1998-10-09 Novel fluorescent reporter molecules and their applications including assays for caspases
BR9814816-8A BR9814816A (pt) 1997-10-10 1998-10-09 Novas moléculas repórter fluorescentes e suas aplicações incluindo análises de caspases
NZ503619A NZ503619A (en) 1997-10-10 1998-10-09 Fluorescent reporter molecules and their applications including assays for caspases
JP2000515498A JP2001519368A (ja) 1997-10-10 1998-10-09 新規の蛍光レポーター分子、およびカスパーゼのアッセイを含むその適用
EP98953317A EP1026988A4 (fr) 1997-10-10 1998-10-09 Nouvelles molecules reporters fluorescentes, applications de ces molecules et dosages des caspases
CA002308125A CA2308125A1 (fr) 1997-10-10 1998-10-09 Nouvelles molecules reporters fluorescentes, applications de ces molecules et dosages des caspases
IL135365A IL135365A0 (en) 1997-10-10 1998-10-09 Fluorescent reporter compounds and methods utilizing the same
KR1020007003886A KR20010031056A (ko) 1997-10-10 1998-10-09 신규 형광 리포터 분자 및 카스파제에 대한 분석을 비롯한이의 용도
HU0100079A HUP0100079A2 (hu) 1997-10-10 1998-10-09 Új, fluoreszcens riportermolekulák és alkalmazásaik, többek között kaszpázok vizsgálatára
EA200000408A EA200000408A1 (ru) 1997-10-10 1998-10-09 Новые флуоресцентные сигнальные молекулы и их применение, в том числе, в анализах каспаз
NO20001322A NO20001322L (no) 1997-10-10 2000-03-14 Nye fluorescerende reportermolekyler og deres anvendelse inkluderende analyser for "kaspaser"
IS5414A IS5414A (is) 1997-10-10 2000-03-24 Nýstárlegar flúrljómandi vísisameindir og notkun þeirra, þ.á.m. til greiningar á kapösum

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US09/033,661 1998-03-03

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WO2000073437A1 (fr) * 1999-05-27 2000-12-07 Merck Frosst Canada & Co. Analyses permettant de detecter l'activite de caspases a l'aide de proteines fluorescentes vertes
WO2001027242A2 (fr) * 1999-10-14 2001-04-19 Saint Louis University Procedes d'identification d'inhibiteurs de methionine aminopeptidases
WO2001036617A2 (fr) * 1999-11-18 2001-05-25 Cyclacel Limited Essai permettant de mesurer l'activite enzymatique in vivo
WO2002012545A2 (fr) * 2000-08-03 2002-02-14 Cytovia, Inc. Procédé d'identification d'agents immunosuppresseurs
WO2002040978A2 (fr) * 2000-10-30 2002-05-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede d'analyse simultanee de deux emissions de fluorescence au moyen d'un cytometre de flux a laser simple
US6716851B2 (en) 2000-12-12 2004-04-06 Cytovia, Inc. Substituted 2-aryl-4-arylaminopyrimidines and analogs as activators or caspases and inducers of apoptosis and the use thereof
US6747052B2 (en) 2000-12-07 2004-06-08 Cytovia, Inc. Substituted indole-2-carboxylic acid benzylidene-hydrazides and analogs as activators of caspases and inducers of apoptosis and the use thereof
US6759207B2 (en) 1997-10-10 2004-07-06 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US6821744B2 (en) 2002-10-29 2004-11-23 Roche Diagnostics Operations, Inc. Method, assay, and kit for quantifying HIV protease inhibitors
WO2005003100A2 (fr) 2003-07-03 2005-01-13 Myriad Genetics, Inc. Composes et leur utilisation therapeutique
US6861419B2 (en) 2000-04-18 2005-03-01 Cytovia, Inc. Substituted 1, 4-thiazepine and analogs as activators of caspases and inducers of apoptosis and the use thereof
US6906203B1 (en) 1999-11-05 2005-06-14 Cytovia, Inc. Substituted 4H-chromene and analogs as activators of caspases and inducers of apoptosis and the use thereof
US6979530B2 (en) 2001-05-21 2005-12-27 Applera Corporation Peptide conjugates and fluorescence detection methods for intracellular caspase assay
US6984718B2 (en) 1998-07-21 2006-01-10 Cytovia, Inc. Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof
US20060140956A1 (en) * 2002-10-11 2006-06-29 Wen-Hwa Lee Method and compounds for inhibiting hec1 ativity for the treatment of proliferative diseases
US7256219B2 (en) 2001-03-14 2007-08-14 Cytovia, Inc. Multifluoro-substituted chalcones and analogs as activators of caspases and inducers of apoptosis and the use thereof
US7304146B2 (en) 2004-01-16 2007-12-04 Applera Corporation Fluorogenic kinase assays and substrates
US7476741B2 (en) 2002-05-16 2009-01-13 Cytovia, Inc. Substituted 4H-chromens, 2H-chromenes, chromans and analogs as activators of caspases and inducers of apoptosis and the use thereof
US7501233B2 (en) 2000-11-10 2009-03-10 Perkinelmer Cellular Technologies Germany Gmbh Method for measuring the vitality of cells
US7528164B2 (en) 2002-05-16 2009-05-05 Cytovia, Inc. Substituted 4-aryl-4h-pyrrolo[2,3-h]chromenes and analogs as activators of caspases and inducers of apoptosis and the use thereof
US7534902B2 (en) 2003-11-14 2009-05-19 Wisconsin Alumni Research Foundation Fluorescence assays with improved sensitivity
US7960384B2 (en) 2006-03-28 2011-06-14 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8084605B2 (en) 2006-11-29 2011-12-27 Kelly Ron C Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
US8222411B2 (en) 2005-09-16 2012-07-17 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8906901B2 (en) 2005-09-14 2014-12-09 Takeda Pharmaceutical Company Limited Administration of dipeptidyl peptidase inhibitors
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US7270801B2 (en) 1997-10-10 2007-09-18 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US6759207B2 (en) 1997-10-10 2004-07-06 Cytovia, Inc. Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof
US6984718B2 (en) 1998-07-21 2006-01-10 Cytovia, Inc. Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof
WO2000054049A3 (fr) * 1999-03-12 2000-12-14 Evotec Analytical Sys Gmbh Mesure de la chimiosensibilite par l'intermediaire de l'activite caspase
WO2000054049A2 (fr) * 1999-03-12 2000-09-14 Evotec Analytical Systems Gmbh Mesure de la chimiosensibilite par l'intermediaire de l'activite caspase
EP1055929A1 (fr) * 1999-05-26 2000-11-29 Tibotec N.V. Moyens et procédé pour la découverte des médicaments et la caractérisation des cellules
US7129036B1 (en) 1999-05-26 2006-10-31 Tibotec Bvba Reporter molecules and methods for assaying the lipidations status of a cell
WO2000073802A1 (fr) * 1999-05-26 2000-12-07 Tibotec Nv Molecules reporteurs et procedes permettant d'evaluer l'activite proteasique a haute specificite
WO2000073497A1 (fr) * 1999-05-26 2000-12-07 Tibotec Nv Molecules rapporteurs et procedes de determination de l'etat d'association de lipides dans une cellule
WO2000073437A1 (fr) * 1999-05-27 2000-12-07 Merck Frosst Canada & Co. Analyses permettant de detecter l'activite de caspases a l'aide de proteines fluorescentes vertes
WO2001027242A3 (fr) * 1999-10-14 2001-12-20 Univ Saint Louis Procedes d'identification d'inhibiteurs de methionine aminopeptidases
US6593454B2 (en) 1999-10-14 2003-07-15 Saint Louis University Methods for identifying inhibitors of methionine aminopeptidases
WO2001027242A2 (fr) * 1999-10-14 2001-04-19 Saint Louis University Procedes d'identification d'inhibiteurs de methionine aminopeptidases
US7507762B2 (en) 1999-11-05 2009-03-24 Cytovia, Inc. Substituted 4H-chromene and analogs as activators of caspases and inducers of apoptosis and the use thereof
US6906203B1 (en) 1999-11-05 2005-06-14 Cytovia, Inc. Substituted 4H-chromene and analogs as activators of caspases and inducers of apoptosis and the use thereof
WO2001036617A3 (fr) * 1999-11-18 2002-05-02 Cyclacel Ltd Essai permettant de mesurer l'activite enzymatique in vivo
WO2001036617A2 (fr) * 1999-11-18 2001-05-25 Cyclacel Limited Essai permettant de mesurer l'activite enzymatique in vivo
US6852906B2 (en) 1999-11-18 2005-02-08 Cyclacel, Ltd. Assay for measuring enzyme activity in vivo
US6861419B2 (en) 2000-04-18 2005-03-01 Cytovia, Inc. Substituted 1, 4-thiazepine and analogs as activators of caspases and inducers of apoptosis and the use thereof
WO2002012545A3 (fr) * 2000-08-03 2002-08-01 Cytovia Inc Procédé d'identification d'agents immunosuppresseurs
WO2002012545A2 (fr) * 2000-08-03 2002-02-14 Cytovia, Inc. Procédé d'identification d'agents immunosuppresseurs
US6828091B2 (en) 2000-08-03 2004-12-07 Cytovia, Inc. Method of identifying immunosuppressive agents
WO2002040978A2 (fr) * 2000-10-30 2002-05-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede d'analyse simultanee de deux emissions de fluorescence au moyen d'un cytometre de flux a laser simple
WO2002040978A3 (fr) * 2000-10-30 2002-11-28 Deutsches Krebsforsch Procede d'analyse simultanee de deux emissions de fluorescence au moyen d'un cytometre de flux a laser simple
US7501233B2 (en) 2000-11-10 2009-03-10 Perkinelmer Cellular Technologies Germany Gmbh Method for measuring the vitality of cells
US6747052B2 (en) 2000-12-07 2004-06-08 Cytovia, Inc. Substituted indole-2-carboxylic acid benzylidene-hydrazides and analogs as activators of caspases and inducers of apoptosis and the use thereof
US7226927B2 (en) 2000-12-12 2007-06-05 Cytovia, Inc. Substituted 2-aryl-4-arylaminopyrimidines and analogs as activators of caspases and inducers of apoptosis and the use thereof
US6716851B2 (en) 2000-12-12 2004-04-06 Cytovia, Inc. Substituted 2-aryl-4-arylaminopyrimidines and analogs as activators or caspases and inducers of apoptosis and the use thereof
US7256219B2 (en) 2001-03-14 2007-08-14 Cytovia, Inc. Multifluoro-substituted chalcones and analogs as activators of caspases and inducers of apoptosis and the use thereof
US6979530B2 (en) 2001-05-21 2005-12-27 Applera Corporation Peptide conjugates and fluorescence detection methods for intracellular caspase assay
US8623995B2 (en) 2001-05-21 2014-01-07 Applied Biosystems, Llc Peptide conjugates and fluorescence detection methods for intracellular caspase assay
US7528164B2 (en) 2002-05-16 2009-05-05 Cytovia, Inc. Substituted 4-aryl-4h-pyrrolo[2,3-h]chromenes and analogs as activators of caspases and inducers of apoptosis and the use thereof
US7476741B2 (en) 2002-05-16 2009-01-13 Cytovia, Inc. Substituted 4H-chromens, 2H-chromenes, chromans and analogs as activators of caspases and inducers of apoptosis and the use thereof
US8252520B2 (en) * 2002-10-11 2012-08-28 Taivex Therapeutics Corporation Methods and compounds for inhibiting Hec1 activity for the treatment of proliferative diseases
US20060140956A1 (en) * 2002-10-11 2006-06-29 Wen-Hwa Lee Method and compounds for inhibiting hec1 ativity for the treatment of proliferative diseases
US6821744B2 (en) 2002-10-29 2004-11-23 Roche Diagnostics Operations, Inc. Method, assay, and kit for quantifying HIV protease inhibitors
WO2005003100A2 (fr) 2003-07-03 2005-01-13 Myriad Genetics, Inc. Composes et leur utilisation therapeutique
US7534902B2 (en) 2003-11-14 2009-05-19 Wisconsin Alumni Research Foundation Fluorescence assays with improved sensitivity
US8034928B2 (en) 2003-11-14 2011-10-11 Wisconsin Alumni Research Foundation Fluorescence assays with improved sensitivity
US7304146B2 (en) 2004-01-16 2007-12-04 Applera Corporation Fluorogenic kinase assays and substrates
US8906901B2 (en) 2005-09-14 2014-12-09 Takeda Pharmaceutical Company Limited Administration of dipeptidyl peptidase inhibitors
US8222411B2 (en) 2005-09-16 2012-07-17 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US7960384B2 (en) 2006-03-28 2011-06-14 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8084605B2 (en) 2006-11-29 2011-12-27 Kelly Ron C Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
EP2875040A1 (fr) * 2012-07-19 2015-05-27 Kingston University Higher Education Corporation Sonde fluorescente à base de rhodamine pour l'activité coagulase et la détection de souches bactériennes produisant de la coagulase
EP2875040B1 (fr) * 2012-07-19 2017-10-18 Kingston University Higher Education Corporation Sonde fluorescente à base de rhodamine pour l'activité coagulase et la détection de souches bactériennes produisant de la coagulase
US10400263B2 (en) 2012-07-19 2019-09-03 Kingston University Higher Education Corporation Tripeptide rhodamine compound
WO2017223147A1 (fr) 2016-06-23 2017-12-28 Life Technologies Corporation Méthodes et compositions de détection ou de mesure de caspases ou d'apoptose
CN107987019A (zh) * 2018-01-08 2018-05-04 深圳市佶达德科技有限公司 一种若丹明双吡唑乙酸酯有机激光材料的制备方法
CN108129388A (zh) * 2018-01-08 2018-06-08 深圳市佶达德科技有限公司 一种若丹明双吡唑乙酸酯有机激光材料及其应用

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KR20010031056A (ko) 2001-04-16
NO20001322D0 (no) 2000-03-14
CA2308125A1 (fr) 1999-04-22
EP1026988A1 (fr) 2000-08-16
PL341661A1 (en) 2001-04-23
AU754634B2 (en) 2002-11-21
EP1026988A4 (fr) 2005-03-30
IL135365A0 (en) 2001-05-20
EA200000408A1 (ru) 2000-12-25
JP2001519368A (ja) 2001-10-23
IS5414A (is) 2000-03-24
CN1281346A (zh) 2001-01-24
NO20001322L (no) 2000-06-13
BR9814816A (pt) 2004-06-22
AU1072299A (en) 1999-05-03
HUP0100079A2 (hu) 2001-05-28

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