WO1999047920A1 - New assays for screening for substances and compounds influencing apoptosis - Google Patents
New assays for screening for substances and compounds influencing apoptosis Download PDFInfo
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- 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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Classifications
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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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Abstract
The invention relates to new assays for detecting substances influencing apoptosis for a new treatment of diseases caused by the human herpesvirus 6 (HHV-6), for example multiple sclerosis.
Description
Neue Assays zum Screenen nach apoptosebeeinflussenden Substanzen und VerbindungenNew assays for screening for apoptosis-influencing substances and compounds
Die vorliegende Erfindung betrifft neue Assays zum Auffinden von apoptose-beein- flussenden Substanzen für eine neue Therapie der durch das humane Herpesvirus 6 (HHV-6) hervorgerufenen Erkrankungen, wie z.B. der multiplen Sklerose (MS) (Eine Übersicht über Erkrankungen durch HHV-6 ist im Artikel D.K.Braun; G. Dominguez, P.E Pellett (1997). Human Herpesvirus 6 Clinical Microbiology Reviews. 10 3 521- 567 zu finden) Neuen Erkenntnissen zufolge wird multiple Sklerose ätiologisch durch Viren, hauptsachlich das humane Herpesvirus Typ 6 (HHV-6) verursacht Das Virus persistiert im Nervengewebe und führt über die Auslosung immunologischer Mechanismen zur Zerstörung des Nervengewebes Der Erfindung liegt die Beobachtung zugrunde, daß eine „death effector domain" (DED)-homologe Region im HHV-6 existiert. Gegenstand der Erfindung sind auf dieser neuen Erkenntnis aufbauende neue Assays zum Auffinden von Substanzen, die diese virale Strategie zum Überleben im infizierten Wirt antagonisieren und zur Beseitigung von HHV-6- infizierten Zellen aus dem Organismus und damit zur Verhinderung immunpathologischer Mechanismen im Nervengewebe führen sowie die in solchen Assays gefundenen Verbindungen, Substanzen und StoffeThe 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. 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
Infektionen des zentralen Nervensystems (ZNS) mit HHV-6 können sowohl bei immundefizienten, als auch bei immunkompetenten Patienten akut oder subakut bis chronisch verlaufen und sind häufig mit diffusen oder multifokalen Demyelinisierungen assoziiert (Carrigan, D R , D Harrington, K K Knox (1996)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)
Subacute leukoencephalitis caused by CNS infection with human herpesvirus-6 manifesting as acute multiple sclerosis Neurology 47 1, 145-148) Bei der Suche nach Pathogenen, die mit multipler Sklerose assoziiert sind, wurden bei über 70% der MS Patienten HHV-6 festgestellt (Challoner, P B , K T Smith, J D Parker, D L MacLeod, S N Coulter, T M Rose, E R Schultz, J L Bennett, R L Garber,Subacute leukoencephalitis caused by CNS infection with human herpesvirus-6 manifesting as acute multiple sclerosis Neurology 47 1, 145-148) When searching for pathogens associated with multiple sclerosis, 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,
M Chang, et al (1995) Plaque-associated expression of human herpesvirus 6 in multiple sclerosis Proc Natl Acad Sei USA 92 16, 7440-7444) Nukleare
- 2 -M Chang, et al (1995) Plaque-associated expression of human herpesvirus 6 in multiple sclerosis Proc Natl Acad Sei USA 92 16, 7440-7444) Nukleare - 2 -
Anfarbungen von HHV-6-Antιgenen in Oligodendrozyten wurde bei MS Patienten festgestellt, nicht jedoch bei Untersuchungsgut von Kontroll-Patienten Bei MS- Patienten wiederum wurde die Anfarbung häufiger in und um Plaques, dagegen nicht bei nicht involvierter weißer Substanz festgestellt Da die Zerstörung von Oligodendrozyten pathognomonisch für MS ist, scheint die Assoziation von HHV-6 mit der Atiologie und/oder der Pathogenese der multiplen Sklerose bewiesenStaining of HHV-6 anti- genes in oligodendrocytes was found in MS patients, but not in the test specimens of control patients. In MS patients, staining was more often found in and around plaques, but not in the case of uninvolved white matter, since the destruction of oligodendrocytes is pathognomonic for MS, the association of HHV-6 with the etiology and / or pathogenesis of multiple sclerosis appears to be proven
Es ist bekannt, daß Viren verschiedene Strategien entwickelt haben, um sich in ihren Wirten zu vermehren und zu überleben Durch die enge Beziehung von Virus und Wιrtszelle/-organιsmus mußte sich ein evolutionäres Wechselspiel mit denIt is known that 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
Abwehrstrategien des Wirtes ergebenDefense strategies of the host result
Einige Viren, z B Influenzaviren oder HIV, produzieren antigene Varianten, um dem Immunsystem des Wirtes zu entkommen Andere, wie z B Adenoviren (AV) oder Zytomegaheviren (CMV), haben immunsuppressive Strategien entwickeltSome viruses, such as influenza viruses or HIV, produce antigenic variants to escape the host's immune system. Others, such as adenoviruses (AV) or cytomegaheviruses (CMV), have developed immunosuppressive strategies
Epstem-Barr Virus, ein Herpesvirus, und Pockenviren wiederum verfügen über Möglichkeiten, mit verschiedenen Zytokinfünktionen zu interferieren Wahrend die genannten Beispiele die infizierte Zelle vor exogener Zerstörung schützen sollen, stellt eine weitere virale Strategie die Verhinderung von Apoptose, einen Schutzmechanismus vor Selbstzerstorung der infizierten Zelle, darEpstem-Barr virus, a herpes virus, and smallpox viruses, in turn, 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
Der programmierte Zelltod eukaryotischer Zellen (Apoptose) ist durch Schrumpfung von Zelle und Kern, Chromatinkondensation und „Blebbing" der Plasmamembran gekennzeichnet Die wahrend dieses Prozesses entstehenden „apoptotic bodies" werden von phagozytierenden Zellen beseitigt Damit unterscheidet sich dieser Vorgang grundlegend vom pathologischen Zelltod, der Nekrose Die Apoptose erfüllt mindestens zwei FunktionenThe programmed cell death of eukaryotic cells (apoptosis) 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
1 Sie ist für die Gewebsdifferenzierung wahrend der Embryogenese und zur1 It is for tissue differentiation during embryogenesis and for
Aufrechterhaltung der Gewebshomoostase notwendig
- 3 -Maintenance of tissue homostasis is necessary - 3 -
2. Sie ist eine von verschiedenen Abwehrstrategien eines multizellulären2. It is one of several defense strategies of a multicellular
Organismus. Die Zelle „erkennt" die potentielle Gefahr, die sie für den Gesamtorganismus im Sinne einer Entartung oder Virusinfektion mit anschließender Vermehrung und Verbreitung des Virus darstellt und „handelt" durch Selbstmord.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.
Der Vorgang „Apoptose" ist somit eine Art zellulärer Altruismus.The "apoptosis" process is therefore a kind of cellular altruism.
Getriggert wird dieser Vorgang unterschiedlich, z.B. durch zytotoxische T-Zel en, durch proinflammatorische Zytokine (wie TNF-α), oder durch virale Störung des zellulären Metabolismus bzw. der Zellzyklusregulation. Diese verschiedenen Induktionsmechanismen münden gemeinsam in der Aktivierung von Mitgliedern einer Familie von Cystein-Proteasen (Caspasen).This process is triggered differently, e.g. by cytotoxic T cells, by pro-inflammatory cytokines (such as TNF-α), or by viral disturbance of cellular metabolism or cell cycle regulation. These different induction mechanisms lead together to the activation of members of a family of cysteine proteases (caspases).
Virale Genprodukte, die Apoptose inhibieren, sind z.B.Viral gene products that inhibit apoptosis are e.g.
• CrmA des Kuhpockenvirus oder• CrmA of the cowpox virus or
• P35 des Baculovirus.• P35 of the baculovirus.
Beide haben direkte Caspase-inhibitorische Wirkung.Both have a direct caspase inhibitory effect.
Sogenannte Bcl-2-homologe virale Proteine, wie z.B.So-called Bcl-2 homologous viral proteins, e.g.
• E1B des Adenovirus oder • BHRF1 des Epstein-Barr Virus• E1B of the adenovirus or • BHRF1 of the Epstein-Barr virus
sind andere Apoptose-Inhibitoren, deren exakter inhibitorischer Mechanismus noch unklar ist.are other apoptosis inhibitors, the exact inhibitory mechanism of which is still unclear.
Kürzlich wurden eine Reihe neuer viraler Proteine beschrieben (S. Hu, C. Vincenz, M.A number of new viral proteins have recently been described (S. Hu, C. Vincenz, M.
Buller, and V.M. Dixit (1997): A novel family of viral death effector domain- containing molecules that inhibit both CD-95-and tumor necrosis factor receptor - 1 -
induced apoptosis J Biol Chem 272, 15 9621-9624, M Thome, P Schneider, K Hofmann, H Fickenscher, E Meinl, F Neipel, C Mattmann, K Burns, J -L Bodmer, M Schroter, C Scaffidi, P H Krammer, M E Peter, and J Tschopp (1997) Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors Nature 386 517-521, J Bertin, R C Armstrong, S Ottihe, D A Martin, Y Wang, SBuller, and VM Dixit (1997): A novel family of viral death effector domain- containing molecules that inhibit both CD-95-and tumor necrosis factor receptor - 1 - induced apoptosis J Biol Chem 272, 15 9621-9624, M Thome, P Schneider, K Hofmann, H Fickenscher, E Meinl, F Neipel, C Mattmann, K Burns, J -L Bodmer, M Schroter, C Scaffidi, PH Krammer, ME Peter, and J Tschopp (1997) Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors Nature 386 517-521, J Bertin, RC Armstrong, S Ottihe, DA Martin, Y Wang, S
Banks, G -H Wang, T G Senkevich, E S Alnemri, B Moss, M J Lenardo, K J Tomaselli, and J I Cohen (1997) Death effector domain-containing herpesvirus and poxvirus proteins mhibit both Fas- and TNFR1 -induced apoptosis Proc Natl Acad Sei USA 94 1 172-1176), die ebenfalls in der Lage sind, die Apoptose infizierter Zellen zu verhindernBanks, G -H Wang, TG Senkevich, ES Alnemri, B Moss, MJ Lenardo, KJ Tomaselli, and JI Cohen (1997) Death effector domain-containing herpesvirus and poxvirus proteins mhibit both Fas- and TNFR1 -induced apoptosis Proc Natl Acad Sei USA 94 1 172-1176), which are also able to prevent the apoptosis of infected cells
• MC 159 des Molluscum contagiosum Virus (MCV),MC 159 of the Molluscum contagiosum Virus (MCV),
• E8 des equinen Herpesvιrus-2 (EHV-2) und• E8 of the equine Herpesvιrus-2 (EHV-2) and
• Proteine in anderen Viren, u a im Kaposi-Sarcom- Virus (HHV-8) und im Herpesvirus saimiπ (HVS)• Proteins in other viruses, including the Kaposi Sarcom virus (HHV-8) and the herpes virus saimiπ (HVS)
Das Wirkprinzip laßt sich folgendermaßen erklaren (siehe auch M Thome, P Schneider, K Hofmann, H Fickenscher, E Meinl, F Neipel, C Mattmann, K Burns, J -L Bodmer, M Schroter, C Scaffidi, P H Krammer, M E Peter, and J Tschopp (1997) Viral FLICE-inhibitory proteins (FLEPs) prevent apoptosis induced by death receptors Nature 386 517-521)The principle of action can be explained as follows (see also M Thome, P Schneider, K Hofmann, H Fickenscher, E Meinl, F Neipel, C Mattmann, K Burns, J -L Bodmer, M Schroter, C Scaffidi, PH Krammer, ME Peter, and J Tschopp (1997) Viral FLICE-inhibitory proteins (FLEPs) prevent apoptosis induced by death receptors Nature 386 517-521)
Die Todes-Rezeptoren (z B Fas=CD95=APO-l), die der TNF-Rezeptor-Superfamilie angehören, kuppeln nach Erhalt des entsprechenden Signals über eine zyto- plasmatische Sequenz, die sogenannte „death domam" (DD) mit homologenThe death receptors (eg Fas = CD95 = APO-1), which belong to the TNF receptor superfamily, couple after receiving the corresponding signal via a cytoplasmic sequence, the so-called "death domam" (DD) with homologous
Adaptoien der Todesmaschinerie (FADD=Fas associated death domain) FADD assoziiert mit einer Interleukin Iß converting enzyme (ICE) - ähnlichen Protease (FLICE, caspase 8, MACH, Mch5) über die sogenannten „death effector domams" (DED), die sowohl am Aminoterminus von FLICE, als auch am Carboxyterminus von FADD vorhanden sind Diese Bindung führt zur Formation des „death inducing signalhng complex" (DISC), der ultimativ zum Ausloser der Apotose wird Gemeinsam ist der neuen Familie von viralen Inhibitoren der Apoptose (vFLIP s), daß
- 5 -Adaptoia of the death machinery (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. This bond leads to the formation of the "death inducing signaling complex" (DISC), which ultimately triggers apotosis. Common to the new family of viral inhibitors of apoptosis (vFLIP s), that - 5 -
sie 2 homologe Regionen zur sogenannten „death effector domain" (DED) der zellulären Mittler der Apoptose besitzen.). Damit sind diese Proteine in der Lage, die Signalkette, die zur Aktivierung der Caspasen führt, durch Interferenz zu unterbrechen.they have 2 homologous regions to the so-called "death effector domain" (DED) of the cellular mediators of apoptosis.) These proteins are thus able to interrupt the signal chain that leads to the activation of the caspases by interference.
Die DED-Homologe stellen außerordentlich interessante antivirale Targets dar, da die Interaktion der Homologen mit entsprechenden zellulären Proteinen sehr spezifisch ist. Damit kann im Gegensatz zu Eingriffen bei direkten Inhibitoren der Caspasen sehr spezifisch therapeutisch interveniert werden. Die Gefahr, durch eine Therapie generalisiert (auch in nicht infizierten Zellen) Apoptose auszulosen, ist demnach nicht gegeben.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).
Bislang sind solche vFLIP's, die homologe Regionen zu DED's zeigen, nur bei den 4 obengenannten Viren, 3 davon sind Herpesviren (EHV-2, HHV-8 und Herpesvirus saimiri) bekannt.So far, such 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).
Es existierte bisher keine Therapie, die diesen Wirkmechanismus bei der Bekämpfung relevanter humanpathogener Viren nutzte.So far, there has been no therapy that uses this mechanism of action in the fight against relevant human pathogenic viruses.
Bei einer Datenbankrecherche wurde auf der Suche nach Homologien zu den DED s des humanen FLICE beim großen Tegumentprotein des HHV-6 eine Übereinstimmung festgestellt'In a database search, a match was found in the search for homologies to the DEDs of the human FLICE with the large tegument protein of the HHV-6 '
FLICE: YDIGEQLDSEDLAS LKFLSLDY IPQRKQEPIKDALMLFQRLQEKRMLEESN LSFL I I I I : 1 I : : I :l : : 1 I : : : I : : I : I : : I : : : : _ : :FLICE: YDIGEQLDSEDLAS LKFLSLDY IPQRKQEPIKDALMLFQRLQEKRMLEESN LSFL I I I I: 1 I:: I: l:: 1 I::: I:: I: I:: I:::: _::
HH\ -6:DDIGSQ - - - QDLVADKTTDLEHAPQKRK- - -KNSHSLE LELNDKKKKDTAS LTYYHH \ -6: DDIGSQ - - - QDLVADKTTDLEHAPQKRK- - -KNSHSLE LELNDKKKKDTAS LTYY
Bei Bindung dieser homologen Region an eine DED wird somit der Tod der infizierten Zelle durch Apoptose gehemmt, das Virus vermehrt sich weiter und führt entweder selbst zu klinischen Symptomen, oder antigenpräsentierende Zellen können kontinuierlich Herpesvirusantigene präsentieren, es kommt zu einer immunologischen Reaktion im ansonsten immunpriveligiertem ZNS.
Die vorliegende Erfindung betrifft jetzt überraschenderweise Assays, die sich mit diesem Wissen konfigurieren lassen sowie Substanzen, die in diesen Assays identifiziert werden und die in infizierten Zellen die Apoptose auslösen.When this homologous region is bound to a DED, the death of the infected cell is inhibited by apoptosis, the virus continues to multiply and either leads to clinical symptoms itself, or antigen-presenting cells can continuously present herpes virus antigens, resulting in an immunological reaction in the otherwise immunogen-activated CNS . Surprisingly, 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.
Die Assays werden wie folgt durchgeführt:The assays are carried out as follows:
Fas-exprimierende Zellen werden mit dem viralen Envelope oder Fragmenten davon transfiziert; diese Zellen können dann keine CD-95 vermittelte Apoptose mehr ausführen. Diese Eigenschaft wird dann folgendermaßen geprüft: die transfiziertenFas-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
Zellen werden trypsiniert und ein Teil in 96-well-Mikrotiterplatten eingesät. Nach einer Zeit von ca. 30 Minuten wird ein agonistischer Fas- Antikörper (Clone CH11; kommerziell erhältlich, z.B. Fa. Clontech) in einer Konzentration von 1 μg/ml Medium hinzugegeben. Das Endvolumen beträgt 100 μl. Die Zellen werden nach weiterer 5 stündigen Inkubation bei 37°C wird das Medium abgesaugt, die Zellen mitCells 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 μg / ml medium. The final volume is 100 μl. After a further 5 hours of incubation at 37 ° C., the cells are suctioned off and the cells are sucked off
PBS gewaschen und ein Annexin- Assay, der kommerziell erhältlich ist (Fa. Clontech) durchgeführt. Die Auswertung erfolgt mittels Fluoreszenzmikroskopie. Nicht apopto- tische Klone werden weiterexpandiert. Der Assay auf apoptoseinduzierende Substanzen wird folgendermaßen durchgeführt: die Zellen werden in 96-well Mikrotiter- Plates oder entsprechend kleinere Formate ausgesät. Die Testsubstanzen werden dazupipettiert. Eine Vorinkubation von 2 Stunden schließt sich an. 100 ng/ml agonistischer anti CD95-Antikörper (Clone CH-11) (oder Fas-Ligand, z.B. Fa. Boeh- ringer Ingelheim) wird hinzugefügt. Nach einer Inkubationszeit von 6 Stunden bis 12 Stunden werden die Zellen mit Kristallviolett gefärbt und im ELISA-Reader quanti- fiziert. Eine alternative Auswertemöglichkeit besteht in Anwendung des kommerziellen Annexin-Assays (Fa. Clontech). Die Quantifizierung würde in diesem Fall über Fluoreszenzdetektoren erfolgen.PBS washed and an 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. After an incubation period of 6 hours to 12 hours, 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.
Die Erfindung beansprucht die Assays, die auf der Interaktion von DED-homologen Regionen des HHV-6 mit humanem FLICE basieren und die darauf aufbauende neuartige antivirale Therapie der durch HHV-6 hervorgerufenen Erkrankungen, z.B. der Multiplen Sklerose mit dem Ziel der Aktivierung der Apoptose in virusinfizierten
- 7 -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 -
Zellen. Sie beansprucht darüberhinaus die in diesen Assays gefundenen Substanzen, Verbindungen und Stoffe, die die Interaktion der viralen DED-homologen Regionen des HHV-6 mit zellulären Partnern hemmen. Solche Substanzen oder Verbindungen können niedermolekulare Verbindungen sein. Sie können auch Peptide sein, die aus der Sequenz der zellulären DED-Domäne bestehen. Diese Peptide können kompetitiv an die virale DED-Domäne binden, diese inaktivieren und so eine Apoptose ermöglichen. Eine mögliche Sequenz wäre z.B.: DIGEQLDSEDLASLKFLSLDYIPQRKQCells. It also claims the substances, compounds and substances found in these assays which inhibit the interaction of the viral DED-homologous regions of the HHV-6 with cellular partners. 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
Im Gegensatz zum derzeitigen Stand der Technik der Therapie der multiplenIn contrast to the current state of the art of multiple therapy
Sklerose, die zumeist immunsuppressiv ausgerichtet ist, hat die neuartige Therapie das Ziel, über die Hemmung der Interaktion von vFLIP's und zellulären DED's selektiv infizierte Zellen durch Apoptose zu eliminieren und chronisch persistente oder latente Infektionen zu verhindern. Hemmt man die Interaktion der vFLIP's und der DED s, beseitigt man darüberhinaus nicht nur die infizierten Zellen, sondert gleichzeitig denSclerosis, 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
Auslöser der Immunreaktion, die in der Pathogenese der multiplen Sklerose eine Rolle spielt.
Triggers the immune response that plays a role in the pathogenesis of multiple sclerosis.
Claims
1. Verfahren zum Screenen von anti HHV-6 wirksamen Substanzen, dadurch gekennzeichnet, daß man Fas-exprimierende Zellen, die mit viralem Tegument oder Fragmenten davon transfiziert sind, mit diesen Substanzen inkubiert und das Auftreten von Apoptose prüft.
1. A method for screening anti-HHV-6 active substances, characterized in that Fas-expressing cells which are transfected with viral tegument or fragments thereof are incubated with these substances and the occurrence of apoptosis is checked.
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| AU33294/99A AU3329499A (en) | 1998-03-19 | 1999-03-09 | New assays for screening for substances and compounds influencing apoptosis |
| JP2000537064A JP2002506995A (en) | 1998-03-19 | 1999-03-09 | Novel assays for screening substances and compounds that affect apoptosis |
| EP99914494A EP1064547A1 (en) | 1998-03-19 | 1999-03-09 | New assays for screening for substances and compounds influencing apoptosis |
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| DE1998112182 DE19812182A1 (en) | 1998-03-19 | 1998-03-19 | Identifying substances with anti-human herpes virus 6 activity useful for treating multiple sclerosis and infections of the central nervous system |
| DE19812182.2 | 1998-03-19 |
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|---|---|---|---|---|
| WO1997009617A1 (en) * | 1995-09-06 | 1997-03-13 | Arch Development Corporation | Screening methods for the identification of inducers and inhibitors of programmed cell death (apoptosis) |
| WO1997012632A1 (en) * | 1995-10-05 | 1997-04-10 | Tkb Associates Limited Partnership | Methods for treatment of diseases associated with a deficiency of fas ligand activity |
-
1998
- 1998-03-19 DE DE1998112182 patent/DE19812182A1/en not_active Withdrawn
-
1999
- 1999-03-09 AU AU33294/99A patent/AU3329499A/en not_active Abandoned
- 1999-03-09 WO PCT/EP1999/001511 patent/WO1999047920A1/en not_active Application Discontinuation
- 1999-03-09 EP EP99914494A patent/EP1064547A1/en not_active Withdrawn
- 1999-03-09 JP JP2000537064A patent/JP2002506995A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997009617A1 (en) * | 1995-09-06 | 1997-03-13 | Arch Development Corporation | Screening methods for the identification of inducers and inhibitors of programmed cell death (apoptosis) |
| WO1997012632A1 (en) * | 1995-10-05 | 1997-04-10 | Tkb Associates Limited Partnership | Methods for treatment of diseases associated with a deficiency of fas ligand activity |
Non-Patent Citations (3)
| 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 * |
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| AU3329499A (en) | 1999-10-11 |
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