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WO1999047920A1 - Nouvelles analyses de filtrage de substances et composes a action apoptose - Google Patents

Nouvelles analyses de filtrage de substances et composes a action apoptose Download PDF

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Publication number
WO1999047920A1
WO1999047920A1 PCT/EP1999/001511 EP9901511W WO9947920A1 WO 1999047920 A1 WO1999047920 A1 WO 1999047920A1 EP 9901511 W EP9901511 W EP 9901511W WO 9947920 A1 WO9947920 A1 WO 9947920A1
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WO
WIPO (PCT)
Prior art keywords
apoptosis
substances
hhv
cells
virus
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Application number
PCT/EP1999/001511
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German (de)
English (en)
Inventor
Olaf Weber
Hubert Hug
Original Assignee
Bayer Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Aktiengesellschaft filed Critical Bayer Aktiengesellschaft
Priority to AU33294/99A priority Critical patent/AU3329499A/en
Priority to JP2000537064A priority patent/JP2002506995A/ja
Priority to EP99914494A priority patent/EP1064547A1/fr
Publication of WO1999047920A1 publication Critical patent/WO1999047920A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to new assays for the detection of apoptosis-influencing substances for a new therapy of the diseases caused by the human herpes virus 6 (HHV-6), such as e.g. multiple sclerosis (MS) (An overview of diseases caused by HHV-6 can be found in the article DKBraun; G. Dominguez, PE Pellett (1997). Human Herpesvirus 6 Clinical Microbiology Reviews. 10 3 521-567) Multiple sclerosis etiologically caused by viruses, mainly the human herpes virus type 6 (HHV-6). The virus persists in the nerve tissue and leads to the destruction of the nerve tissue via the triggering of immunological mechanisms.
  • HHV-6 multiple sclerosis
  • the invention is based on the observation that a "death effector domain" (DED ) -Homologous region exists in HHV-6.
  • the subject of the invention are new assays based on this new finding for the detection of substances which antagonize this viral strategy for survival in the infected host and for the removal of HHV-6-infected cells from the organism and thus leading to the prevention of immunopathological mechanisms in nerve tissue and the compounds found in such assays, p substances and substances
  • Central nervous system (CNS) infections with HHV-6 can be acute or subacute to chronic in both immunodeficient and immunocompetent patients and are often associated with diffuse or multifocal demyelination (Carrigan, D R, D Harrington, K K Knox (1996)
  • HHV-6 Subacute leukoencephalitis caused by CNS infection with human herpesvirus-6 manifesting as acute multiple sclerosis Neurology 47 1, 145-148)
  • HHV-6 was found in over 70% of MS patients ( Challoner, PB, KT Smith, JD Parker, DL MacLeod, SN Coulter, TM Rose, ER Schultz, JL Bennett, RL Garber,
  • viruses have developed various strategies in order to multiply and survive in their hosts.
  • the close relationship between virus and host cell / organism required an evolutionary interplay with the
  • viruses such as influenza viruses or HIV
  • AV adenoviruses
  • CMV cytomegaheviruses
  • Epstem-Barr virus, a herpes virus, and smallpox viruses have the ability to interfere with various cytokine functions. While the examples mentioned are intended to protect the infected cell from exogenous destruction, another viral strategy is the prevention of apoptosis, a protective mechanism against self-destruction of the infected cell , represents
  • the programmed cell death of eukaryotic cells is characterized by shrinkage of the cell and nucleus, chromatin condensation and "blebbing" of the plasma membrane.
  • the "apoptotic bodies” that arise during this process are eliminated by phagocytic cells. This process differs fundamentally from pathological cell death, necrosis Apoptosis fulfills at least two functions
  • Organism The cell "recognizes" the potential danger it poses to the whole organism in the sense of a degeneracy or virus infection with subsequent multiplication and spread of the virus and "acts" by suicide.
  • the "apoptosis” process is therefore a kind of cellular altruism.
  • cytotoxic T cells e.g. by cytotoxic T cells, by pro-inflammatory cytokines (such as TNF- ⁇ ), or by viral disturbance of cellular metabolism or cell cycle regulation.
  • pro-inflammatory cytokines such as TNF- ⁇
  • viral disturbance of cellular metabolism or cell cycle regulation e.g. by cytotoxic T cells, by pro-inflammatory cytokines (such as TNF- ⁇ ), or by viral disturbance of cellular metabolism or cell cycle regulation.
  • cysteine proteases caspases
  • Viral gene products that inhibit apoptosis are e.g.
  • So-called Bcl-2 homologous viral proteins e.g.
  • FADD Fas associated death domain
  • FADD associated with an interleukin ß converting enzyme (ICE) - like protease (FLICE, caspase 8, MACH, Mch5) via the so-called “death effector domams” (DED), which are both on Amino terminus from FLICE, as well as at the carboxy terminus of FADD are present.
  • ICE interleukin ß converting enzyme
  • DED death effector domams
  • This bond leads to the formation of the “death inducing signaling complex” (DISC), which ultimately triggers apotosis.
  • DISC death inducing signaling complex
  • DED death effector domain
  • the DED homologs are extremely interesting antiviral targets because the interaction of the homologues with the corresponding cellular proteins is very specific. In contrast to interventions with direct caspase inhibitors, this can be used to intervene therapeutically in a very specific manner. There is therefore no danger of triggering apoptosis through therapy (even in non-infected cells).
  • vFLIPs that show homologous regions to DEDs are only known for the 4 viruses mentioned above, 3 of which are herpes viruses (EHV-2, HHV-8 and herpes virus saimiri).
  • HH ⁇ -6 DDIGSQ - - - QDLVADKTTDLEHAPQKRK- - -KNSHSLE LELNDKKKKDTAS LTYY
  • the present invention now relates to assays which can be configured with this knowledge and substances which are identified in these assays and which trigger apoptosis in infected cells.
  • the assays are carried out as follows:
  • Fas-expressing cells are transfected with the viral envelope or fragments thereof; these cells can then no longer carry out CD-95-mediated apoptosis. This property is then checked as follows: the transfected
  • Cells are trypsinized and some of them are sown in 96-well microtiter plates. After a time of approximately 30 minutes, an agonistic Fas antibody (Clone CH11; commercially available, e.g. from Clontech) is added in a concentration of 1 ⁇ g / ml medium. The final volume is 100 ⁇ l. After a further 5 hours of incubation at 37 ° C., the cells are suctioned off and the cells are sucked off
  • annexin assay which is commercially available (from Clontech) performed.
  • the evaluation is carried out using fluorescence microscopy. Non-apoptotic clones are expanded further.
  • the assay for apoptosis-inducing substances is carried out as follows: the cells are sown in 96-well microtiter plates or correspondingly smaller formats. The test substances are pipetted in. A pre-incubation of 2 hours follows. 100 ng / ml agonistic anti CD95 antibody (clone CH-11) (or Fas ligand, e.g. from Boehringer Ingelheim) is added.
  • the cells are stained with crystal violet and quantified in the ELISA reader.
  • An alternative evaluation option is to use the commercial annexin assay (from Clontech). In this case, the quantification would be carried out using fluorescence detectors.
  • the invention claims the assays which are based on the interaction of DED-homologous regions of the HHV-6 with human FLICE and the novel antiviral therapy based thereon of the diseases caused by HHV-6, for example multiple sclerosis with the aim of activating apoptosis in virus infected - 7 -
  • Such substances or compounds can be low molecular weight compounds. They can also be peptides consisting of the sequence of the cellular DED domain. These peptides can competitively bind to the viral DED domain, inactivate it and thus enable apoptosis.
  • a possible sequence would be, for example: DIGEQLDSEDLASLKFLSLDYIPQRKQ
  • Sclerosis which is mostly immunosuppressive, aims to eliminate selectively infected cells by apoptosis by inhibiting the interaction of vFLIPs and cellular DEDs and to prevent chronic persistent or latent infections. If you inhibit the interaction of the vFLIPs and the DEDs, you not only eliminate the infected cells, but also the cells

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne de nouvelles analyses permettant de détecter des substances à action apoptose pour un nouveau traitement des maladies dues à l'herpèsvirus humain 6 (HHV-6), telles que la sclérose multiple.
PCT/EP1999/001511 1998-03-19 1999-03-09 Nouvelles analyses de filtrage de substances et composes a action apoptose WO1999047920A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU33294/99A AU3329499A (en) 1998-03-19 1999-03-09 New assays for screening for substances and compounds influencing apoptosis
JP2000537064A JP2002506995A (ja) 1998-03-19 1999-03-09 アポトーシスに作用する物質および化合物をスクリーニングするための新規アッセイ
EP99914494A EP1064547A1 (fr) 1998-03-19 1999-03-09 Nouvelles analyses de filtrage de substances et composes a action apoptose

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1998112182 DE19812182A1 (de) 1998-03-19 1998-03-19 Neue Assays zum Screenen nach apoptosebeeinflussenden Substanzen und Verbindungen
DE19812182.2 1998-03-19

Publications (1)

Publication Number Publication Date
WO1999047920A1 true WO1999047920A1 (fr) 1999-09-23

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PCT/EP1999/001511 WO1999047920A1 (fr) 1998-03-19 1999-03-09 Nouvelles analyses de filtrage de substances et composes a action apoptose

Country Status (5)

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EP (1) EP1064547A1 (fr)
JP (1) JP2002506995A (fr)
AU (1) AU3329499A (fr)
DE (1) DE19812182A1 (fr)
WO (1) WO1999047920A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19962978C1 (de) * 1999-12-24 2001-08-30 Aloys Wobben Windenergieanlage mit einem turmgestützten Maschinenkopf

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997009617A1 (fr) * 1995-09-06 1997-03-13 Arch Development Corporation Procede de criblage d'inducteurs et d'inhibiteurs de la mort programmee des cellules (apoptose)
WO1997012632A1 (fr) * 1995-10-05 1997-04-10 Tkb Associates Limited Partnership Methodes de traitement de maladies associees a une deficience de l'activite du ligand fas

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997009617A1 (fr) * 1995-09-06 1997-03-13 Arch Development Corporation Procede de criblage d'inducteurs et d'inhibiteurs de la mort programmee des cellules (apoptose)
WO1997012632A1 (fr) * 1995-10-05 1997-04-10 Tkb Associates Limited Partnership Methodes de traitement de maladies associees a une deficience de l'activite du ligand fas

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; DOCKRELL D H ET AL: "Human herpesvirus 6.", XP002108737, retrieved from STN Database accession no. 1999166886 *
KIRN, EKKEHARD ET AL: "In vitro cytobiological effects of human herpesviruses 6 and 7: immunohistological monitoring of apoptosis, differentiation and cell proliferation", ANTICANCER RES. (1997), 17(6D), 4623-4632, XP002108736 *
MAYO CLINIC PROCEEDINGS, (1999 FEB) 74 (2) 163-70. REF: 56 *

Also Published As

Publication number Publication date
EP1064547A1 (fr) 2001-01-03
JP2002506995A (ja) 2002-03-05
DE19812182A1 (de) 1999-09-23
AU3329499A (en) 1999-10-11

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