[go: up one dir, main page]

WO1999066065A2 - Activite de proteasomes - Google Patents

Activite de proteasomes Download PDF

Info

Publication number
WO1999066065A2
WO1999066065A2 PCT/GB1999/001840 GB9901840W WO9966065A2 WO 1999066065 A2 WO1999066065 A2 WO 1999066065A2 GB 9901840 W GB9901840 W GB 9901840W WO 9966065 A2 WO9966065 A2 WO 9966065A2
Authority
WO
WIPO (PCT)
Prior art keywords
proteasomal
assay
protein
tat
viral
Prior art date
Application number
PCT/GB1999/001840
Other languages
English (en)
Other versions
WO1999066065A3 (fr
Inventor
Hans-Peter Schmid
Franck Petit
Peter-Michael Kloetzel
Anne-Sophie Jarrousse
Karine Gautier
Saloua Badaoui
Said Mouzeyar
Paul Nicolas
Original Assignee
Zarpex Biosciences Limited
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
Priority claimed from GBGB9812756.6A external-priority patent/GB9812756D0/en
Priority claimed from GBGB9812759.0A external-priority patent/GB9812759D0/en
Priority claimed from GBGB9812758.2A external-priority patent/GB9812758D0/en
Priority claimed from GBGB9812760.8A external-priority patent/GB9812760D0/en
Priority claimed from GBGB9812757.4A external-priority patent/GB9812757D0/en
Application filed by Zarpex Biosciences Limited filed Critical Zarpex Biosciences Limited
Priority to JP2000554874A priority Critical patent/JP2002518020A/ja
Priority to CA002330210A priority patent/CA2330210A1/fr
Priority to EP99957075A priority patent/EP1088100A2/fr
Priority to AU42811/99A priority patent/AU4281199A/en
Publication of WO1999066065A2 publication Critical patent/WO1999066065A2/fr
Publication of WO1999066065A3 publication Critical patent/WO1999066065A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • 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
    • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
    • C12Q2337/20—Coumarin derivatives
    • C12Q2337/22—7-Amino-4-methylcoumarin, i.e. AMC, MCA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/163—Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • proteolytic system decreases the concentrations of specific proteins and their attendant activities.
  • the specificity of this process is achieved by a highly selective mechanism, mediated by large, multi-subunit cytosolic complexes, the proteasomes [1].
  • Proteasomes can also degrade ribonucleic acid, and are particularly effective against viral and cytokine mRNAs [2].
  • the major proteolytic activity in the cell is the 26S proteasome, a key regulatory protease which controls physiological processes as diverse as the cell cycle [3], apoptosis [4], oncoprotein degradation [5], gene expression [6], the inflammatory response [6] and bulk proteolysis [7].
  • the proteasome consists of a catalytic core (20S) to which is attached regulatory complexes [8].
  • the cylindrical catalytic core comprises 28 subunits — two copies each of seven different ⁇ -type and seven different ⁇ -type subunits — assembled to form a stack of four seven-membered rings with the general structure ⁇ ]_7 ⁇ _7 ⁇ _7 ⁇ _7.
  • This stack contains internal chambers harbouring the proteolytic threonine residues. Regulatory complexes (19S), each containing 6 ATPases and at least 9 other subunits, are attached to the proteasome complex.
  • An 1 IS regulatory complex substitutes for the 19S regulator [9] when the proteasome fragments antigens for MHC Class I presentation.
  • selective interchange of both core and regulatory subunits also occurs in response to various physiological stimuli (for example, exposure to interferon- ⁇ ) [4, 10,11].
  • proteasome The diverse functions of the proteasome depend on multiple catalytic sites which include chymotrypsin-like, trypsin-like and peptidylglutamylpeptide hydrolysing activities. Unlike other proteolytic systems, however, these proteasomal activities are energy-dependent.
  • Ubiquitin is a protein of 76 amino acid residues. Its function is as a 'tag' for proteins which are to be degraded by the 26S proteasome. This 'tagging' involves the covalent attachment of multiple chains of ubiquitin to the protein. Ubiquitin is first activated by a ubiquitin-activating enzyme (El). Ubiquitin-conjugating enzymes (E2s), with the help of ubiquitin-protein ligases (E3s), then join several (4-12) ubiquitins sequentially to a lysine residue in the target protein, leading to the formation of multi-ubiquitin chains [12].
  • El ubiquitin-activating enzyme
  • E2s Ubiquitin-conjugating enzymes
  • E3s ubiquitin-protein ligases
  • Multi-ubiquitinylated proteins are first recognised by subunits of the 19S regulator [13] which presents the protein chain for processing within the 20S cylindrical core.
  • Other 19S regulator subunits can recognise non-ubiquitinylated proteins.
  • Ubiquitinylated and non-ubiquitinylated proteins cannot enter the proteolytic chambers of the 20S particle without being unfolded in a complex process requiring the chaperone functions of the 19S regulator [14].
  • Concomitant with unfolding and degradation, multi-ubiquitin chains are removed from target proteins by proteasome- associated ubiquitin carboxy-terminal hydrolases (UCHs); the ubiquitin is recycled [15].
  • UCHs proteasome- associated ubiquitin carboxy-terminal hydrolases
  • UCHs are members of a large family of ubiquitin-specific cysteine proteases, many of which are free in the cytosol [16]. proteins are continuously ubiquitinylated and de- ubiquitinylated in the cell in an editing process controlled by E3s and cytosolic uchs. multi-ubiquitinylated proteins failing the inspection process and remaining multi- ubiquitinylated, such a oxidation-damaged proteins or proteins of viral origin, are degraded by the 26s proteasome. This degradation process can also be activated or inhibited by large (130-250 kda) cytosolic regulatory proteins [17].
  • Proteasomes are large multi-unit protease complexes that play a key role in the control of cellular processes by selectively degrading intracellular proteins.
  • Tagged proteins are recognised by elements within the 19S proteasomal regulator, unfolded, de-ubiquitinylated and translocated to the internal chamber of the 20S proteasomal core complex for partial or complete hydrolysis by an array of proteolytic functionalities.
  • the process may be subject to further control by cytosolic regulators.
  • NF- ⁇ B The active form of the protein NF- ⁇ B is required for the expression of a large number of genes involved in immune and inflammatory responses; these genes encode inflammatory and chemotactic cytokines, haematopoietic growth factors, cell adhesion molecules, antibodies, class I MHC molecules and cytokine receptors, as well as the key enzymes nitric oxide synthetase and cyclooxygenase-2.
  • NF- ⁇ B is normally inactive in the cytosol; it is activated by the proteasome pathway in response to a variety of pathogenic stimuli, including viruses, bacteria, radiation, oxidants and inflammatory cytokines. Inhibitors directed against this specific proteasomal function should therefore provide therapeutic benefit [18] in a wide range of inflammatory and allergic diseases such as rheumatoid arthritis, inflammatory bowel diseases, and asthma.
  • the cellular immune system acts naturally to defend the body from foreign proteins. This aggressive response is the major reason why transplanted tissues suffer rejection. It is also inappropriately elicited by 'self proteins in autoimmune disease (multiple sclerosis, lupus erythrematosus, etc.) This response is initiated by the degradation of non-recognised proteins into short antigenic peptide fragments by 20S/1 IS proteasome complexes, and by the presentation of these on the cell surface with the almost ubiquitous MHC-I antigen. Inhibitors of the formation of these antigens would, therefore, be potentially useful in tissue transplantation and in the treatment of autoimmune conditions.
  • the cycle of reactions involved in cell division is controlled by cyclin-dependent protein kinases.
  • the activity of particular kinase complexes during different phases of the cell cycle is regulated by the amount of specific proteins in the cell, this amount being controlled by the balance between synthesis and proteasome-dependent degradation [19].
  • proteasomal catalytic function leads to cessation of cell growth and may induce apoptosis. It is reasonable therefore to expect that appropriate inhibitors may have utility in the treatment of a variety of cancers and non-malignant hyperproliferative conditions such as psoriasis and restenosis.
  • inhibitors More recently developed inhibitors are indanone-containing peptides [21], glyoxals [22] and further boronic acids [23].
  • proteasomes The relationships between the 20S proteasome, the 20S + 19S (26S) complex, the 1 IS (PA28) complex, the MHC class I, cell division and general cellular proteolysis are well established.
  • the relationships between proteasomal complexes and RNA are often considered to be non-specific.
  • 20S proteasomes contain small RNA [24,25] and interfere with protein synthesis [26,27].
  • proteasomes contain an endonuclease activity which selectively degrades RNAs [28]. This selectivity is expressed against RNAs with AU-rich elements [29]. Such sequences are found in retroviral RNAs.
  • the nuclease activity is associated with subunits zeta and iota in the 20S proteasome [2].
  • the proteasome has an essential antiviral role in vivo. Viral proteins synthesised in infected cells are partially degraded by the proteasome [1-3]. Peptides so generated are bound to MHC class-I molecules and presented on the cell surface where they are recognised by cytotoxic T-lymphocytes. Viruses have developed mechanisms which enable them to subvert this process [4].
  • HIV RNA codes for a number of proteins which interfere with proteasome function.
  • Vpu and Env act together to increase the proteasome-mediated degradation of CD4, important for adequate functioning of helper T cells [5].
  • Ne binds to the B-subunit Hs ⁇ 3 [6] although the function of this binding remains unknown. This subunit is also the binding site for Tax-1, a protein encoded by HTLV [7].
  • Viral proteins in naive cells are degraded by the proteasome — probably by the 20S complex alone, without ubiquitinylation. If this degradation is blocked by inhibitors, p24 Gag proteins accumulate in the cytoplasm and more proviral DNA is synthesised [8]. Tat binds to the 20S proteasome, and strongly inhibits the proteolytic activity, as well as blocking the formation of the 20S-1 IS complex [9].
  • Tat occurs in the naive cell either as a result of synthesis de novo or passage from neighbouring infected cells, a process facilitated by the presence in its sequence of an RGD domain which allows binding to cell surface proteins and consequent cell entry.
  • Tat binds to the TAR of the viral RNA, protects the RNA from degradation by the proteasomal endonuclease and allows effective transcription to proceed.
  • Tat binds to the proteasome and blocks the proteolytic destruction of viral proteins, thereby preventing presentation of viral antigens on the cell surface. Inhibition of Tat binding to the proteasome or of subsequent T ⁇ t-associated events will therefore allow the proteasome to degrade essential viral proteins and to present peptides derived from these on MHC class-I molecules for cellular destruction by cytotoxic lymphocytes.
  • HTLV-I Tax protein associates with the HsN3 subunit. Tax may then act as an anchor for processing of I ⁇ B (to which Tax also binds) and thus be driver of the mechanism whereby NF- ⁇ B is activated by this virus [7].
  • the Hepatitis B virus X protein HBX binds to the subunit XAPC7 and this interaction is important for HSX-mediated transactivation in vivo [10].
  • the Human Papilloma Virus E7 oncoprotein binds to both the retinoblastoma tumour suppressor protein, Rb, and to the S4 ATPase, a subunit of the 19S regulator of the proteasome; this binding is linked to the proteolysis of Rb by the proteasome [11].
  • Another 19S ATPase, SUG1 (S8), is stimulated by specific mRNA sequences [12].
  • Typical examples of the sequences of these viral proteins which affect proteasomal function are shown by way of example in Table 1. [SEQ ID Nos. 1,2,3]. Table 1
  • the invention is designed to identify compounds which will inhibit viral replication and pathogenesis following infection of living cells by the class of viruses whose genomes code for proteins or nucleic acids that bind to the proteasome or cause the binding of host proteins or nucleic acids to the proteasome such that the functioning of the proteasome in cellular surveillance is impeded.
  • the invention consists of an assay which contains proteasomal protein (20S proteasomes separately with or without 19S and 1 IS complexes), viral gene product, and protein or peptide substrates for detection of proteolytic activity by fluorescence, light absorption, luminescence, radioactivity or other physical parameter.
  • the assay may contain viral ribo- or deoxyribo-nucleic acid or an oligonucleotide containing sequences recognised by proteasomal subunits or other host proteins binding to proteasomal subunits; nuclease activity is measured spectrophotometrically, fluorometrically, radiometrically or by chemiluminescence.
  • Both types of assay may in addition contain host proteins which bind virally-encoded protein or nucleic acid such that proteasomal proteolytic or nuclease activities are modulated.
  • Inhibitor compounds, inhibitory amino acid sequences, and medicinal compositions derived therefrom are also considered to form part of the present invention. Where legally permissible the invention also provides a method of treatment of viral disease in an infected patient by administration of an inhibitor obtained according to the assay of the invention.
  • Multiwell (96 or 384 wells per plate) streptavidin coated black plates for example, Reacti- bindTM NeutravidinTM Coated polystyrene plates, pkg of 5 ref. 15117 from Pierce) are stored dessicated at 4°C.
  • wash buffer 25mM-TrisHCl, 150mM-NaCl, 0.05%(v/v) Tween® 20, pH 7.6.
  • substrate solution containing 25pmol of a 31-mer oligoribonucleotide containing one or more AUUUA sequences and tagged with fluorescein at the 5 '-end and biotin at the 3 '-end. Plates are then incubated for 16h at room temperature before each well is washed with 200 ⁇ l of wash buffer.
  • TBK160 buffer (20mM-TrisHCl, 160mM-KCl, 5mM-MgCl2, 3mM-dithiothreitol, pH 7.4).
  • TBK160 containing compounds to be tested or equivalent vehicle
  • 20S proteasomes are purified by standard methods and stored at 4° in TBK600 (20mM-TrisHCl, 600mM-KCl, 5mM-MgCl2, 3mM-dithiothreitol 5 pH 7.4, to which is added 5mM-NaN3 as preservative).
  • This solution is diluted with TBKO (20mM-TrisHCl, 5mM-MgCl2, 3mM-dithiothreitol 5 pH 7.4) so that the final concentration is that of TBK160.
  • HBV X-protein is obtained by purification from extracts of Escherichia coli containing the HBV X-protein gene and appropriate expression vector. It is stored in PsP buffer (50mM-Na phosphate, lOOmM-NaCl, ImM-EDTA, ImM-dithiothreitol, pH 7.0).
  • Control wells for proteasomal RNase activity contain the same as above but without HBV X-protein.
  • Controls for HBV X-protein RNase activity contain the same as above but without proteasomal protein.
  • Standard multiwell (96 or 384 wells per plate) plates are used. To each well are added 50 ⁇ l of buffer (30mM-TrisHCl, lOmM-KCl, 5mM-MgCl2, 0.5mM-dithiothreitol, pH 7.8), containing compounds to be tested or equivalent vehicle. At this stage the plates are ready for incubation with assay solutions.
  • 20S proteasomes are purified by standard methods and stored at 4° in TBK600 (20mM-TrisHCl, 600mM-KCl, 5mM-MgCl2, 3mM-dithiothreitol 5 pH 7.4, to which is added 5mM-NaN3 as preservative).
  • This solution is diluted with TBKO (20mM-TrisHCl, 5mM-MgCl2, 3mM-dithiothreitol 5 pH 7.4) so that the final concentration is that of TBK160.
  • HBV X-protein is obtained by purification from extracts of Escherichia coli containing the HBV X-protein gene and appropriate expression vector. It is stored in PsP buffer (50mM-Na phosphate, lOOmM-NaCl, lmM-EDTA, ImM-dithiothreitol, pH 7.0).
  • Proteasome and HBV X-protein solutions are mixed and incubated at 37° for 20min, such that 50 ⁇ l of the resulting mixture contain lpmol of proteasomal protein and 2pmol of HBV X-protein.
  • Control wells for proteasomal protease activity contain the same as above but without HBV X-protein.
  • Controls for HBV X-protein protease activity contain the same as above but without proteasomal protein.
  • Fluorescent product is only released after cleavage of the substrate peptide. Plates incubated with buffer alone will show little or no hydrolysis and readings will be low. With proteasomes alone, cleavage of the substrate takes place and readings are high. When HBV X-protein is present, cleavage is inhibited and readings will therefore be lower than with proteasomal protein alone. The extent to which the effect of HBV X-protein is overcome is a measure of the efficacy of test compounds.
  • the invention relates to a method for identifying compounds which change the concentrations of cytokines, lymphokines and other regulatory proteins by modulating the rate of proteasomal destruction of the specific messenger ribonucleic acids.
  • E-selectin endothelial-leukocyte adhesion molecule- 1
  • VCAM-1 vascular cell adhesion molecule- 1
  • IAM-1 intercellular adhesion molecule- 1
  • IKB is tagged for degradation by ubiquitinylation after phosphorylation of two specific serine residues stimulated by extracellular cytokines.
  • concentration of circulating cytokines is determined by the amount of specific messenger RNA in the originator cells. An increase in the proteasomal degradation of this mRNA will lead to a decrease in the cytokine concentration in the blood and consequently decreases in free NFKB and expression of cellular adhesion proteins.
  • Compounds which modulate proteasomal nuclease activity may therefore have utility in the treatment of inflammatory disease.
  • proteasome exhibits an endonuclease activity which has specificity for the RNA substrate [1].
  • proteasomal RNase is highly effective against AU-rich elements containing two or more AUUUA sequences [SEQ ID No. 4] in the 3*-UTR of the mRNA.
  • AUUUA multimers have an essential role in decreasing the stability and translational efficiency of cytokine mRNAs [3].
  • Insertion of multimeric AUUUA destabilises ⁇ -globin mRNA [4-6] is essential for glucocorticoid stimulation of turnover of interferon- ⁇ mRNA [7] and is responsible for inhibition of the translation of TNF ⁇ -mRNA [8].
  • TNF ⁇ induces phosphorylation of two specific serines of I ⁇ B- ⁇ (which is also constitutively phosphorylated). Inhibition of this phosphorylation is anti-inflammatory because NFKB, the factor needed for increased expression of adhesion molecules is not released from its complex with I ⁇ B- ⁇ [9]. In addition to its effects on the synthesis adhesion molecules, NFKB affects expression of IL-1, IL-6 and TNF- ⁇ [10]. Cytokines also increase the production of reactive oxygen species [11, 12] which may be important regulators of NFKB [reviewed in 13]. Supported by many anti-oxidants inhibiting cytokine-induced phosphorylation of I ⁇ B- ⁇ [10, 11,14-17].
  • Cytokine messenger RNAs differ from the majority of other mRNAs in having several AUUUA sequences in their UTR. This sequence is recognised by the 20S proteasome. This permits the development of a novel method for identifying compounds which change the amount of specific mRNAs without having an effect on the general population of mRNAs.
  • the invention consists of an assay which contains proteasomal protein (20S proteasomes separately with or without 19S and 1 IS complexes), a synthetic oligonucleotide with a 3 '-region containing one or more AUUUA recognition sequences and with or without specific proteins which regulate nuclease activity through binding to one or more of the subunits of the 20S proteasome or of the 19S or 1 IS complexes.
  • Nuclease activity is measured spectrophotometrically, fluorometrically, radiometrically or by chemiluminescence.
  • Compounds are identified by changes in the rate of the cleavage of the oligonucleotide by the proteasomal endonuclease. This may be because the compound binds to the AUUUA sequence or sequences in the oligonucleotide, to one or more of the 20S proteasome or of the 19S or 1 IS complexes, or to the regulatory protein.
  • the invention also provides for a method of treatment of a patient having an inflammatory disease by administration of a compound obtained according to the assay of the invention.
  • Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule- 1 in endothelial cells stimulated to generate radicals. Arterioscler. Thromb. 14, 1665-1673.
  • VCAM-1 Vascular cell adhesion molecule- 1
  • HIV Human Immunodeficiency Virus
  • AIDS Acquired ImmunoDeficiency Syndrome
  • Sufferers from AIDS usually die from infection with opportunistic organisms normally readily resisted in the uninfected population.
  • AIDS is currently treated with a 'triple therapy' of a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor and a protease inhibitor. This triple therapy has been developed because of the virus' remarkable ability to mutate rapidly and hence become resistant to monotherapy.
  • HIV RNA encodes relatively few proteins that are targets for antiviral chemotherapy.
  • Reverse transcriptase and the aspartyl protease have been the major foci for drug development to date.
  • the protein Tat which comprises 86 amino acids and is crucial to viral replication within the host cell, is an attractive target for inhibition but work on this has been slow because of the lack of a robust high-throughput assay.
  • a few compounds have been developed but have not progressed to the clinic, largely because they are peptidic and have poor bioavailability and pharmacokinetic profiles. These have been developed using assay techniques which rely on the direct interaction of Tat with the TAR (transactivation response region) of the viral RNA.
  • Tat In addition to binding to the TAR region of the viral RNA, Tat binds to subunits of the 20S and 19S components of the proteasome. This binding inhibits the proteasomal endonuclease activity and hence permits transcription and replication of the virus.
  • an assay which allows the identification of compounds which interfere with the action of Tat on both RNA and the proteasomal complexes. Previous assay systems do not contain the proteasome or any of its components and therefore cannot detect binding to the proteasome. This novel assay will be of use in the identification of novel compounds with utility in the treatment of AIDS in HIV infected patients.
  • the invention provides an assay which consists of a synthetic oligonucleotide with a TAR sequence upstream of two or more AUUUA sequences, a proteasomal preparation having nuclease activity and comprising any or all of the 20S, 19S and 1 IS components and HIV Tat or a polypeptide containing the sequence of amino acids 48 to 57 of HIV Tat and/or sequences involved in the binding to the 20S, 19S or 1 IS proteasomal complexes.
  • Nuclease activity is measured spectrophotometrically, fluorometrically, radiometrically or by chemiluminescence.
  • Nuclease activity is decreased in the presence of Tat or polypeptides which bind to the TAR of the added oligonucleotide or to one or more subunits of the 20S, 19S or 1 IS proteasomal complexes.
  • Compounds which bind to Tat, the added polypeptides, the TAR of the added oligonucleotide or proteasomal subunits mediating the inhibition of nuclease activity such that nuclease activity in the assay system is increased are identified as Tat inhibitors and will block replication of HIV in infected cells. Such compounds are potentially of value in the treatment of HIV infection.
  • the invention also provides a kit which comprises the components mentioned above necessary for carrying out an assay for identifying Tat inhibitors. Also the invention provides a method for identifying one or more Tat inhibitors) by use of the assay or kit. Tat inhibitor compounds, inhibitory amino acid sequences, and medicinal compositions derived therefrom (whether peptidic or peptidomimetic) useful in title treatment of AIDS in HIV infected patients and obtained or obtainable by the use of the method, assay or kit are also considered to form part of the present invention. Where legally permissible the invention also provides for a method of treatment of AIDS in an HIV infected patient by administration of a Tat inhibitor.
  • a buffer solution containing a synthetic oligonucleotide e.g. SEQ ID 5, 5'CUGGUUAGACCAGAUCUGAGCCUGGGAGC UCUCUGGCUAACUAGAGGAUGC AUUUAUUUAUUAUUUUAGCG ' , known as TARAU4
  • a synthetic oligonucleotide e.g. SEQ ID 5, 5'CUGGUUAGACCAGAUCUGAGCCUGGGAGC UCUCUGGCUAACUAGAGGAUGC AUUUAUUAUUAUUUAGCG ' , known as TARAU4
  • biotin binds to streptavidin such that the oligonucleotide is bound to each well and remains so after washing.
  • Reaction is initiated by addition of a preparation of 20S proteasomes isolated from human spleen. After a fixed time interval, plates are washed. Nuclease activity cleaves the substrate oligonucleotide such that the fluorescent label is released into the medium and removed by washing. Thus proteasomes will, in the absence of Tat, degrade the oligonucleotide and little fluorescence will be detected still bound to each well after washing. In the presence of Tat, nuclease activity will be inhibited and greater fluorescence will remain in each well after washing. Compounds which inhibit the effect of Tat are therefore detected by decreases in the fluorescence left in wells after washing in assays containing proteasomes and Tat.
  • Multiwell (96 or 384 wells per plate) streptavidin coated black plates for example, Reacti- bindTM NeutravidinTM Coated polystyrene plates, pkg of 5 ref. 15117 from Pierce) are stored dessicated at 4°C.
  • wash buffer 25mM-TrisHCl, 150mM-NaCl, 0.05%(v/v) Tween® 20, pH 7.6.
  • substrate solution containing 25pmol of a 31-mer oligoribonucleotide containing one or more AUUUA sequences and tagged with fluorescein at the 5'-end and biotin at the 3'-end. Plates are then incubated for 16h at room temperature before each well is washed with 200 ⁇ l of wash buffer.
  • TBK160 buffer (20mM-TrisHCl, 160mM-KCl, 5mM-MgCl2 and, pH 7.4).
  • TBK160 containing compounds to be tested or equivalent vehicle
  • 20S proteasomes are purified by standard methods and stored at 4° in TBK600 (20mM-TrisHCl, 600mM-KCl, 5mM-MgCl2, 3mM-dithiothreitol 5 pH 7.4, to which is added 5mM-NaN3 as preservative).
  • This solution is diluted with TBKO (20mM-TrisHCl, 5mM-MgCl2, 3mM-dithiothreitol ⁇ pH 7.4) so that the final concentration is that of TBK160.
  • Tat is obtained by purification from extracts of Escherichia coli containing the Tat gene and appropriate expression vector. It is stored in PsP buffer
  • Proteasome and Tat solutions are mixed and incubated at 37° for 20min, such that 50 ⁇ l of the resulting mixture contain lpmol of proteasomal protein and 2pmol of Tat.
  • Control wells for proteasomal RNase activity contain the same as above but without Tat.
  • Controls for Tat RNase activity contain the same as above but without proteasomal protein.
  • proteasome a ubiquitous cellular organelle, has an essential antiviral role in vivo. Viral proteins synthesised in infected cells are partially degraded by the proteasome [30, 31, 32]. Peptides so generated are bound to MHC class-I molecules are presented on the cell surface where they are recognised by cytotoxic T-lymphocytes. Viruses have developed mechanisms which enable them to subvert this process [33].
  • HIV RNA codes for a number of proteins which interfere with proteasome function.
  • Vpu and Env act together to increase the proteasome-mediated degradation of CD4, important for adequate functioning of helper T cells [34].
  • Ne binds to the B-subunit HsN3 [35] although the function of this binding remains unknown. This subunit is also the binding site for Tax-1, a protein encoded by HTLV [36].
  • Viral proteins in naive cells are degraded by the proteasome — probably by the 20S complex alone, without ubiquitinylation. If this degradation is blocked by inhibitors, p24 Gag proteins accumulate in the cytoplasm and more proviral DNA is synthesised [37]. Tat binds to the 20S proteasome, and strongly inhibits the proteolytic activity, as well as blocking the formation of the 20S-1 IS complex [38].
  • Tat occurs in the na ⁇ ve cell either as a result of synthesis de novo or passage from neighbouring infected cells, a process facilitated by the presence in its sequence of an RGD domain which allows binding to cell surface proteins and consequent cell entry.
  • Tat binds to the TAR of the viral RNA, protects the RNA from degradation by the proteasomal endonuclease and allows effective transcription to proceed.
  • Tat binds to the proteasome and blocks the proteolytic destruction of viral proteins, thereby preventing presentation of viral antigens on the cell surface. Inhibition of Tat binding to the proteasome or of subsequent Tat-associated events will therefore allow the proteasome to degrade essential viral proteins and to present peptides derived from these on MHC class-I molecules for cellular destruction by cytotoxic lymphocytes.
  • the HIV protein Tat binds to subunits of the 20S and 19S components of the proteasome. This binding inhibits the proteasomal protease activity. By so doing, Tat helps to maintain the amounts of viral proteins necessary for transcription and replication of the virus and decreases the presentation of virally-derived peptides to the immune system.
  • Previous assay systems for identification of compounds affecting Tat function are based on the binding of Tat to the TAR of viral RNA, and do not contain the proteasome or any of its components. Thus they cannot detect binding to the proteasome.
  • the invention consists of an assay method which allows the identification of compounds which interfere with the inhibition of proteasomal function by the HIV Tat protein.
  • the assay contains proteasomal protein (20S proteasomes separately with or without 19S and 1 IS complexes), Tat protein (or a partial sequence of Tat which contains those regions of the protein that interact with proteasomal subunits) and protein or peptide substrates as appropriate for detection of proteolytic activity by measurement of fluorescence, absorption, luminescence or radioactivity.
  • Protease activity is decreased in the presence of Tat or peptides binding to the Tat binding sites to the 20S or 19S components of the proteasomal system.
  • Compounds which bind to Tat or proteasomal subunits mediating the inhibition of protease activity such that protease activity in the assay system is increased are identified as Tat inhibitors and will block replication of HIV in infected cells. This novel assay will be of use in the identification of compounds with utility in the treatment of AIDS in infected patients.
  • the invention also provides a kit which comprises the components mentioned above necessary for carrying out an assay for identifying Tat inhibitors. Also the invention provides a method for identifying one or more Tat inhibitor(s) by use of the assay or kit.
  • Tat inhibitor compounds, inhibitory amino acid sequences, and medicinal compositions derived therefrom (whether peptidic or peptidomimetic) useful in the treatment of AIDS in HIV infected patients and obtained or obtainable by the use of the method, assay or kit are also considered to form part of the present invention.
  • the invention also provides for a method of treatment of AIDS in an HIV infected patient by administration of a Tat inhibitor obtained according to the invention.
  • Standard multiwell (96 or 384 wells per plate) plates are used. To each well are added 50 ⁇ l of buffer (30mM-TrisHCl, lOmM-KCl, 5mM-MgCl2, 0.5mM-dithiothreitol, pH 7.8), containing compounds to be tested or equivalent vehicle. At this stage the plates are ready for incubation with assay solutions.
  • 20S proteasomes are purified by standard methods and stored at 4° in TBK600 (20mM-TrisHCl, 600mM-KCl, 5mM-MgCl2, 3mM-dithiothreitol s pH 7.4, to which is added 5mM-NaN3 as preservative).
  • This solution is diluted with TBKO (20mM-TrisHCl, 5mM-MgCl2, SmM-dithiothreitol ⁇ pH 7.4) so that the final concentration is that of TBK160.
  • Tat is obtained by purification from extracts of Escherichia coli containing the Tat gene and appropriate expression vector. It is stored in PsP buffer
  • Proteasome and Tat solutions are mixed and incubated at 37° for 20min, such that 50 ⁇ l of the resulting mixture contain lpmol of proteasomal protein and 2pmol of Tat.
  • Control wells for proteasomal protease activity contain the same as above but without Tat.
  • Controls for Tat protease activity contain the same as above but without proteasomal protein.
  • Fluorescent product is only released after cleavage of the substrate peptide. Plates incubated with buffer alone will show little or no hydrolysis and readings will be low. With proteasomes alone, cleavage of the substrate takes place and readings are high. When Tat is present, cleavage is inhibited and readings will therefore be lower than with proteasomal protein alone. The extent to which the effect of Tat is overcome is a measure of the efficacy of test compounds.
  • Bacterial and viral diseases of plants and non-human animals are sources of major economic cost. Crops may fail to germinate or thrive, and storage during transportation to market or by the end-consumer is often limited by such infections. In horticulture, such diseases may, in addition, be the cause of blemishes and misshapen growth. The high standards now set for meat production demand that the animals involved are free of infection at the time of killing. Weight gain in young animals of agricultural importance may also be severely compromised by infectious disease. Increased resistance, particularly of a generic kind, can help to prevent these costly infections.
  • proteasome is a ubiquitous organelle which exhibits an endonuclease activity with specificity for its RNA substrate [1].
  • proteasomes destabilise sequences with AU-rich elements containing two or more AUUUA repeats in the UTR of the mRNA and may be identical with the RNase E-like activity reported by Wennborg et al. [3].
  • AU-rich elements are found in mRNAs of both RNA and DNA viruses and are zones which are sensitive to RNase attack. Such sequences are uncommon in eukaryotic mRNAs [2,3].
  • the endonuclease activity of the proteasome is associated with two ⁇ -type subunits, zeta and iota; of these, zeta has the greater activity [5].
  • the purified zeta subunit which is soluble, retains the endonuclease activity and its selectivity for the AU-rich sequence. It degrades the RNA from Tobacco Mosaic Virus (TMV) but neither 5S ribosomal RNA nor globin mRNA [1].
  • TMV Tobacco Mosaic Virus
  • a typical sequence of the proteasomal zeta subunit is shown by way of example in Table 2.
  • the invention consists of the transfection of the gene for the proteasomal zeta subunit into the host genome (as, for example in plants, in [6]) and its expression under the control of a specific promoter which may be general (see [7]), or inducible by addition of exogenous compounds (see [8]).
  • a specific promoter which may be general (see [7]), or inducible by addition of exogenous compounds (see [8]).
  • zeta subunit is synthesised in the cytoplasm and destroys viral and bacterial RNA containing the recognition and cleavage site (AU-rich) sequences, hence generating resistance to the consequences of infection, preventing further spread of the causative organism.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • AIDS & HIV (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Rheumatology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Pain & Pain Management (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne la modulation et/ou la régulation de l'activité de protéasomes. L'invention concerne des méthodes diagnostiques d'identification de composés inhibant une réplication virale et une pathogenèse, une méthode d'identification d'inhibiteurs de la fonction inhibitrice de nucléase de la protéine TAT du virus de l'immunodéficience humaine, des méthodes d'identification d'inhibiteurs de la fonction inhibitrice de protéase de la protéine TAT du virus de l'immunodéficience humaine, des méthodes d'identification de composés qui modifient les concentrations de protéines régulatrices en modulant la vitesse de destruction de protéasomes d'ARN messagers spécifiques, et des méthodes de génération d'une résistance aux lésions dues à une infection bactérienne ou virale. L'invention concerne des méthodes diagnostiques, des trousses d'application de ces méthodes diagnostiques, des composés identifiés par ces méthodes diagnostiques, ainsi que des séquences d'acides aminés codant ces composés et des compositions médicamenteuses dérivées de ces composés. Lorsque la loi le permet, l'invention concerne également des méthodes de traitement sur la base de la modulation/régulation de l'activité de protéasomes.
PCT/GB1999/001840 1998-06-13 1999-06-10 Activite de proteasomes WO1999066065A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000554874A JP2002518020A (ja) 1998-06-13 1999-06-10 プロテアソーム活性
CA002330210A CA2330210A1 (fr) 1998-06-13 1999-06-10 Activite de proteasomes
EP99957075A EP1088100A2 (fr) 1998-06-13 1999-06-10 Activite de proteasomes
AU42811/99A AU4281199A (en) 1998-06-13 1999-06-10 Proteasomal activity

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
GB9812758.2 1998-06-13
GB9812757.4 1998-06-13
GBGB9812756.6A GB9812756D0 (en) 1998-06-13 1998-06-13 Assay procedure for identifying inhibitors of the nuclease-inhibitory function of the human immunodeficiency virus tat protein
GBGB9812759.0A GB9812759D0 (en) 1998-06-13 1998-06-13 A procedure for generating resistance to bacterial or viral infection damage
GBGB9812758.2A GB9812758D0 (en) 1998-06-13 1998-06-13 Proteasomal destruction of mRNA
GB9812756.6 1998-06-13
GBGB9812760.8A GB9812760D0 (en) 1998-06-13 1998-06-13 A method for identifying compounds which inhibit viral replication and pathogenesis
GB9812759.0 1998-06-13
GBGB9812757.4A GB9812757D0 (en) 1998-06-13 1998-06-13 Assay procedure for identifying inhibitors of the protease-inhibitory function of the human immunodeficiency virus tat protein
GB9812760.8 1998-06-13

Publications (2)

Publication Number Publication Date
WO1999066065A2 true WO1999066065A2 (fr) 1999-12-23
WO1999066065A3 WO1999066065A3 (fr) 2000-04-20

Family

ID=27517463

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1999/001840 WO1999066065A2 (fr) 1998-06-13 1999-06-10 Activite de proteasomes

Country Status (5)

Country Link
EP (1) EP1088100A2 (fr)
JP (1) JP2002518020A (fr)
AU (1) AU4281199A (fr)
CA (1) CA2330210A1 (fr)
WO (1) WO1999066065A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1123412B1 (fr) * 1998-10-20 2004-10-06 Millennium Pharmaceuticals, Inc. Procede de controle de l'action medicamenteuse de l'inhibiteur de proteasome

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587300A (en) * 1994-04-26 1996-12-24 Wisconsin Ulumni Research Foundation Method to increase regulatory molecule production
US5693483A (en) * 1996-01-05 1997-12-02 Icos Corporation Cytoplasmic modulators of integrin binding/signalling
US5932425A (en) * 1997-02-18 1999-08-03 Signal Pharmaceuticals, Inc. Compositions and methods for modulating cellular NF-κB activation
EP0943624A1 (fr) * 1998-03-12 1999-09-22 Universiteit Utrecht Inhibiteurs peptidiques de la rétrorégulation du récepteur de l'hormone de croissance

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1123412B1 (fr) * 1998-10-20 2004-10-06 Millennium Pharmaceuticals, Inc. Procede de controle de l'action medicamenteuse de l'inhibiteur de proteasome

Also Published As

Publication number Publication date
WO1999066065A3 (fr) 2000-04-20
AU4281199A (en) 2000-01-05
CA2330210A1 (fr) 1999-12-23
EP1088100A2 (fr) 2001-04-04
JP2002518020A (ja) 2002-06-25

Similar Documents

Publication Publication Date Title
Cesaro et al. Inhibition of PKR by Viruses
Kim et al. Emerging roles of desumoylating enzymes
Mermoud et al. Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing
Kawai et al. Toll‐like receptor and RIG‐1‐like receptor signaling
Han et al. Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes
Gale Jr et al. Translational control of viral gene expression in eukaryotes
Nair et al. Endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis E virus
Yalamanchili et al. Cleavage of transcriptional activator Oct-1 by poliovirus encoded protease 3Cpro
Zong et al. The functional deubiquitinating enzymes in control of innate antiviral immunity
Hara et al. Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site
Honig et al. Crimean–Congo hemorrhagic fever virus genome L RNA segment and encoded protein
Been et al. Self‐cleaving ribozymes of hepatitis delta virus RNA
Raines Ribonuclease a
Matthews et al. Carboxypeptidase N: a pleiotropic regulator of inflammation
Taylor et al. Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain
Gillette et al. The 19S complex of the proteasome regulates nucleotide excision repair in yeast
Chen et al. Degradation of p21cip1 in cells productively infected with human cytomegalovirus
D'Angelo et al. Cathepsin H is an additional convertase of pro-granzyme B
Duclair et al. High-affinity RNA aptamers against the HIV-1 protease inhibit both in vitro protease activity and late events of viral replication
Lindner Deubiquitination in virus infection
Chen et al. Power and promise of ubiquitin carboxyl-terminal hydrolase 37 as a target of cancer therapy
Rubinstein et al. Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter
WO1999066065A2 (fr) Activite de proteasomes
Karasik et al. The unusual role of ribonuclease L in innate immunity
JPH11137252A (ja) Ｃ型肝炎ウイルスのｎｓ３プロテアーゼを阻害するｒｎａ分子

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 1999957075

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2330210

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 42811/99

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2000/07445

Country of ref document: ZA

Ref document number: 200007445

Country of ref document: ZA

WWP Wipo information: published in national office

Ref document number: 1999957075

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 09719171

Country of ref document: US

WWW Wipo information: withdrawn in national office

Ref document number: 1999957075

Country of ref document: EP