WO2000075310A1 - Interleukin-1-receptor associated kinase-3 (irak3) - Google Patents

Interleukin-1-receptor associated kinase-3 (irak3) Download PDF

Info

Publication number
WO2000075310A1
WO2000075310A1 PCT/EP2000/005290 EP0005290W WO0075310A1 WO 2000075310 A1 WO2000075310 A1 WO 2000075310A1 EP 0005290 W EP0005290 W EP 0005290W WO 0075310 A1 WO0075310 A1 WO 0075310A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
seq
set forth
il
according
Prior art date
Application number
PCT/EP2000/005290
Other languages
French (fr)
Other versions
WO2000075310A8 (en
Inventor
Filippo Volpe
Original Assignee
Glaxo Group 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 to GB9913180.7 priority Critical
Priority to GBGB9913180.7A priority patent/GB9913180D0/en
Application filed by Glaxo Group Limited filed Critical Glaxo Group Limited
Publication of WO2000075310A1 publication Critical patent/WO2000075310A1/en
Publication of WO2000075310A8 publication Critical patent/WO2000075310A8/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

There is disclosed a novel interleukin-1 receptor associated kinase (IRAK). Isolated forms, variants, cDNA, antibodies, modulators, medicaments, methods of treatment and drug screening methods are also disclosed.

Description

INTERLEUKIN-1 -RECEPTOR ASSOCIATED KINASE-3 (IRAK3)

The present invention relates to interieukin-1 receptor associated protein kinases (IRAKs) and their use in drug screening. The present invention also relates to modulators of IRAKs, medicaments, e.g. pharmaceutical compositions, comprising the modulator and methods of treating diseases or conditions affected by IL-1 family activity, e.g. aberrant IL-1 family activity.

lnterleukin-1 (IL-1 ) is a pleiotropic cytokine involved in regulation of host immune responses to pro-inflammatory stimuli. IL-1 induces a wide spectrum of local and systemic changes, some of which are mediated via novel synthesis of pro-inflammatory molecules and cytokines (1 ,2). The induction of pro-inflammatory genes is often mediated by activation of members of the NF- KB family of transcription factors. In unstimulated cells, NF-κB is associated with an inhibitory molecule termed l B and is localised in the cytoplasm (25). In response to IL-1 stimulation lκB is phosphorylated and rapidly degraded. NF-κB, now free from its inhibitory partner, translocates to the nucleus activating transcription.

Similarity between the intracellular portion of IL-1 Rl and the Drosophila Toll receptor, involved in dorsal-ventral patterning (3) and immunity (4), suggest that both receptors could share functional similarity. Indeed Toll signalling activates Pelle kinase inducing phosphorylation of Cactus and activation/transiocation of Dorsal to the nucleus; both proteins are homologous of lκB and NF-κB respectively.

Recently new components involved in the signal transduction cascade leading to the activation of NF-κB have been identified, reviewed in (5,6). Briefly, following IL-1 binding to the IL-1 R1 an IL-1 accessory protein (IL- 1 RAcP) (7-9), MyD88 (10-12) and an IRAK (13-15) are rapidly recruited to the IL-1/IL-R1 complex. IRAK, the mammalian homologue of Drosophila Pelle becomes phosphorylated upon recruitment to the IL-1 receptor complex and is consequently released from the complex (1 1 ). Recently another putative kinase (IRAK2) similar to IRAK has been described (16). IRAK2 preferentially interacts with the cytopiasmic domain of IL-1 R1 whereas IRAK preferentially interacts with the intracellular domain of IL-1 RAcP. The role of IRAK2 in IL-1 signal transduction remains unclear. However, evidence that IL-1 induced NF-kB activation and gene expression are impaired in IRAK-deficient ceils points to an important role for the kinase in IL-1 signal transduction. A novel TRAF family member, TRAF6 (17) has also described as a signal transducer molecule for IL-1 signalling. There is no evidence for the recruitment of TRAF6 to the IL-1 R1 complex however it is clear that TRAF6 acts downstream of IRAK connecting IRAK to NF-κB inducing kinase, NIK (18). Indeed, TRAF6 interacts with and activates NIK (19), causing activation of the IKK complex (20-22) resulting in the phosphorylation of lκB and the translocation of NF-κB to the nucleus.

Due to its pivotal role in the genesis of inflammation, the IL-1 transduction system is an attractive target for therapeutic intervention. A naturally occuring antagonist (IL-1 Ra) to the IL-1 receptor (IL-R) is known to exist however its therapeutic use may be limited due to the necessity to administer large amounts. Up to 100 to 1000 fold amounts of IL-1 Ra greater than IL-1 agonists seem necessary to inhibit biological activities. (24)

We therefore reasoned that novel molecules related to the key signalling components in the IL-1 pathway may also be important targets for intervention in this pathway.

In accordance with the present invention, there is provided an isolated mammalian protein comprising the amino acid sequence as set forth in SEQ.I.D.NO:1 or SEQ.I.D.NO:3 or a variant thereof. Preferably the amino acid sequence is as set forth in SEQ.l.D.NO:3. The present invention provides a novel IRAK protein ( herein "IRAK3") comprising a death domain (amino acids 23 to 106 of SEQ.I.D.NO:1 ) at the amino terminal end followed by a kinase domain (amino acids 155 to 453) and a carboxyl terminal domain (amino acids 454 to 597). Homologous analysis indicates that IRAK3 death domain primary amino acid sequence has 23% identity with the death domain of IRAK whereas IRAK3 kinase domain primary amino acid sequence has 40% identity with IRAK kinase domain.

The term "isolated" as used herein is intended to mean that the protein or nucleic acid as described herein exists in a physical milieu distinct from that in which it occurs in nature. For example, the protein or nucleic acid may be isolated with respect to one or more materials it is associated with in the natural state. It is preferred that proteins and nucleic acid sequences are provided in purified form, preferably at least 50%, more preferably at least 75% e.g. 90%, 95% and most preferably at least 98% pure.

Included within the ambit of the present invention are variants of the protein as set forth in SEQ.I.D.NO-1 or SEQ.I.D.NO:3. Such variants include fragments, analogues, derivatives and splice variants. The term " variant" refers to a protein or part of a protein that retains substantially the same biological function or activity as the protein as set forth in SEQ.I.D.NO:1 or SEQ.I.D.NO:3.

Fragments can include a part of SEQ I.D. NO:1 or SEQ.I.D.NO:3 which retains sufficient identity to the original protein to be effective for example in a screen for identification of modulators of IRAK3 function or activity. Thus fragments include the death domain, kinase domain and carboxyl terminal domain as identified herein. Fragments or the entire protein may be fused to other amino acids or proteins or may be comprised within a larger protein. Such fragment or entire protein may be comprised within a precursor protein designed for expression in a host. Fragments also include alpha helix or alpha-helix forming regions, beta-sheet and beta-sheet forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, amiphipathic regions, flexible regions, substrate binding regions and regions of high antigenic index.

Derivatives include naturally occurring allelic variants. An allelic variant is an alternative form of a protein sequence which may have a substitution, deletion or insertion of one or more amino acids, which does not substantially alter the function of the protein. Derivatives can also be non-naturally occurring proteins or fragments in which one or more of the amino acid sequence as set forth in SEQ.I.D.NO:1 , SEQ.l.D.NO:3 or fragment thereof has been substituted, deleted, rearranged or modified yet biological activity or function or its effectiveness in a screen is substantially retained ( i.e. a conservative change). Furthermore, non-naturally occuring derivatives include proteins or fragments wherein one or more amino acids have been added or inserted into the amino acid sequence as set forth in SEQ.I.D.NO:1 or SEQ.I.D.NO:3 yet biological activity or function or its effectiveness in a screen is substantially retained ( i.e. a conservative change). It will be appreciated by those skilled in the art that various amino acids may be substituted or interchanged for other amino acids without substantially altering certain properties of the protein. The classification of amino acids are typically arranged into groups such as amino acids with aromatic side chains, basic side chains, amide side chains and sulphur containing side chains groups. Within these groups, certain amino acids are interchangable without substantially affecting the function or activity of the resulting protein compared to the unchanged protein.

Also encompassed within the present invention are proteins or fragments which have at least 70% identity to the sequence as set forth in SEQ.I.D.NO:1 or SEQ.I.D.NO:3, preferably at least 80%, more preferably at least 90% e.g. 91 %,92%,93%,94%,95%,96%,97%,98% or even 99% identity. The sequence set forth in SEQ.I.D.NO:1 or SEQ.l.D.NO:3 is derivable from human however, also encompassed within the present invention are other mammalian species variants ( and fragments thereof), particularly primate, rat and mouse. By the term "derivable" it is intended to convey a source not only in the sense of it being the physical source for the material but also to define material which has structural and/or functional characteristics which correspond to those materials but which does not orginate from the reference source.

Analogues include precursor proteins which may be converted by cleavage of the precursor portion to the mature form of the protein.

The protein of the present invention may be recombinant protein, a natural protein or a synthetic protein, preferably a recombinant protein. The protein is recovered and purified from recombinant cell cultures by methods known to those skilled in the art, including ammonium sulfate or ethanol precipitation, acid extraction, gel filtration anion or cation exchange chromatography, phosphocellulose chromatography and lectin chromatography. Protein refolding steps may be used, as necessary, in completing configuration of the mature protein. Finally, HPLC may be employed in the final purification steps.

The polynucleotide of SEQ.I.D.NO:2 codes for the protein having the amino acid sequence of SEQ.I.D.NO:3.

In a further aspect of the present invention, there is provided a polynucleotide sequence comprising the sequence as set forth in SEQ.I.D.NO:2. A different coding sequence may be provided which takes into account the known redundancy or degeneracy of the genetic code. In preferred embodiments, SEQ.I.D.NO:2 further comprises a start triplet codon capable of being recognised by RNA polymerase to thereby initiate transcription of the polynucleotide sequence when incorporated into an appropriate vector within a host cell. Such vectors maybe chromosomal or non-chromosomal, preferably non-chromosomal vectors include plasmids, minichromosomes, transposons or phage, preferably plasmids. Incorporation into a suitable vector may be achieved according to methods well-known to those skilled in the art, see for example Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Spring Harbor, NY 1989 . It is preferred that the vector further comprises one or more regulatory elements capable of directing mRNA synthesis, e.g. a promoter operatively linked to the sequence, a ribosome binding site for translation initiation and transcription terminator. The vector may also comprise a selectable marker, such markers are well known to those skilled in the art. Host cells transformed with vectors of the present invention may be prokaroytic or eukaryotic, preferably a higher eukaryotic cell such as a mammalian cell, e.g. HEK293T and HeLa cells.

Alternatively, a cell free translation system may be employed to produce protein from RNA derived from DNA vectors according to the present invention.

The present invention further provides a protein encoded by the polynucleotide of SEQ.I.D.NO:2. The invention further provides the polynucleotide which codes for the protein having amino acid sequence of SEQ.I.D.N0.3.

Nucleotide sequences which hybridise to SEQ.I.D.NO:2 are also provided in the present invention. Hybridisation is preferably under stringent conditions. One example is where attempted hybridisation is carried out at a temperature of from 35°c to about 65°c using a salt solution which is about 0.9mo!ar. However, the skilled person is readily able to vary such conditions in order to take into account variables such as base composition, types of ions present etc.

Also provided are complementary or anti-sense strands of the sequence of SEQ.I.D.N0.2. Modified forms of the sequence of SEQ.I.D.NO:2 or fragments thereof may be used in gene therapy strategies to downregulate the expression of IRAK3. Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a nucleotide sequence to DNA or RNA.

In other aspects of the present invention, there is also provided antibodies capable of binding to the protein of SEQ.I.D.NO:1 or SEQ.l.D.NO-3. Such antibodies may be monoclonal or polyclonal and may be chimaeric or humanised according to methods well known to those skilled in the art. Also included are fragments thereof, e.g. F(ab')2 fragments, Fab fragments, Fv fragments, ScFv fragments and CDR peptides.

The present invention also extends to methods of identifying compounds capable of modulating IRAK3 function or activity. Such methods are known or apparent to those skilled in the art. For example, measuring the incorporation of 32P into IRAK3 from γ32-ATP in a standard kinase assay in the presence of the candidate agent over control is one method available to those skilled in the art although other assays will be readily apparent.

The present invention provides modulators, e.g. antagonists of IRAK3 function or activity and also medicaments comprising the same. Preferably antagonists are provided in pharmaceutically acceptable purity. Antagonists of the present invention may act by disrupting or perturbing the function or activity of one or more of the domains of IRAK3 i.e. the carboxy terminal domain, kinase domain or death domain of the protein of SEQ.I.D.NO:1 or SEQ.I.D.NO:3. More particularly, antagonists may inhibit the phosphorylation of IRAK3 by its upstream regulator, for example, inhibiting phosphorylation of the specific threonine at position 69 of IRAK3. This may be achieved through binding of the antagonist to IRAK3, particularly to position 69, so as to prevent phosphorylation or by direct disruption of the enzymatic activity of the phosphorylating kinase. Other options include disrupting the interaction of IRAK3 with either its downstream and/or upstream substrates/interactive partners.

Therefore the invention provides a modulator e.g. antagonist of a protein or variant thereof as described above identifiable by a method described above for use in therapy. The invention further provides the use of a modulator of the protein as identified above optionally identifiable by the method described above in the manufacture of a medicament for the treatment of diseases or conditions affected by IL-1 family activity. Medicaments of the present invention are typically pharmaceutical compositions comprising an effective amount of the modulator, a pharmaceutically acceptable carrier or diluent and optionally another therapeutic agent. Compositions may be in a form suitable for parenteral administration i.e. subcutaneously, intramuscularly or intravenously but depending on the nature of the antagonist other routes such as oral, inhalation, intra-nasal, topical or intra articular may be more appropriate. Compositions for parenteral administration will commonly comprise a solution of the antagonist or cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used e.g. water, buffered water, 0.4% saline and the like. These solutions are sterile and generally free of particulate matter. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like. Medicaments of the present invention may comprise other therapeutic agents.

The invention further provides a method of treating a mammalian patient, including prophylatically where appropriate, a disease or condition affected by IL-1 family activity (e.g. IL-1 a, IL-1 b, IL-1g or IL-18, IL-1 delta, see EP 0879889A2 and human Toll receptors) especially excessive or deleterious IL- 1 family activity comprising the step of administrating an effective amount of a modulator as described herein. An effective amount, i.e. therapeutically effective amount of the modulator may be determined by methods known or apparent to those skilled in the art such as dose escalation studies. Treatment protocol will depend on a number of considerations including the severity of the condition to be treated, the weight and age of the patient, such considerations being within the purview of the attending physician. Medicaments of the present invention may be used in conjunction with e.g. simultaneously, separately or sequentially with other anti-inflammatory agents. Also provided are medicaments, e.g. pharmaceutical compositions comprising a therapeutically effective amount of the protein having a primary amino acid sequence as set forth in SEQ.I.D.NO:1 , SEQ.I.D.NO:3 or variant thereof in the manufacture of a medicament for the treatment of diseases or conditions particularly those affected by IL-1 family activity especially excessive or deleterious activity.

In accordance with a further aspect of the present invention, there is provided the use of a modulator of the protein as set forth in SEQ.I.D.NO:1 ,

SEQ.I.D.NO:3 or variant thereof in the manufacture of a medicament for the treatment of inflammatory disorders and diseases.

Diseases or conditions that may be treated with medicaments of the present invention include inflammatory pathologies ( both acute and chronic), postoperative septicemia, autoimmune diseases such as inflammatory bowel disease ( Crohn's disease and ulcerative Colitis), psoriasis, multiple sclerosis, transplant rejection and graft versus host disease, stroke, ischaemia reperfusion injury, acute respiratory disease syndrome, restenosis, atherosclerosis, AIDS, bone diseases such as osteoporosis ( e.g. post menstrual or osteoporosis of disuse), cancer, Alzheimers disease, rheumatoid arthritis, poor wound healing and asthma.

The present invention is illustrated with reference to the following examples An IRAK3 protein was identified and sequenced. Motif analysis highlighted the presence of a typical death domain, a kinase domain and carboxyl terminal domain.

IRAK3 Expression Constructs

A pcDNA-amp (Invitrogen) was modified by introducing a FLAG epitope

(DYDDDDK) downstream of an optimal start codon (pCMV-FLAG).

Oligonucleotides: 5'-AGCTTCCACCATGGACTACAAGGACGACGATGACAAAGTGG-3'

(SEQ.I.D.NO: 12) and

5AAATTCCACTTTGTCATCGTCGTCCTTGTAGTCCATGGTGA-3, antisense (SEQ.I.D.NO: 4) were annealed and subcloned into pcDNA-amp Hindlll/EcoRI digested. Full length IRAK3 was subcloned into this digested vector as an EcoRI - Notl fragment.

IRAK3-Death Domain (IRAK-3DD) was generated by PCR using the following primers: 5'-CGGAATTCGGCGCACACGCTGCTGTTCGACCTGC-3' (SEQ-I.D.NO-5)

5'-GCTCTAGATCTATTGAAAGCTGATGGAAGATTTAAGCAG-3'

(SEQ.I.D.NO.6)

The PCR fragment was digested with EcoRI - Xbal and subcloned into pCMV-

FLAG EcoRI - Xbal digested and fully sequenced. Reporter gene pNF- B luc as described in ref 12 (Bums et al). These reporter gene constructs are transiently transfected into HEK293 cells as described below.

Full length IRAK cDNA (amino acids 1-712) was cloned as an EcoRI-Xbal fragment into pcDNA-amp (invitrogen) (pCMV-IRAK) as described in ref.9 IRAK2 and TRAF6-dominant negative cDNAs were obtained by RT-PCR from messenger RNA from human skeletal muscle (Clontech) using the following oiigonucleotide pairs:

RECTIFIED SHEET (RULE 91) ISA/EP '-CGAATTCGCCTGCTACATCTACCAGCTGCCC-3' (IRAK2-1 ), (SEQ.I.D.NO:7) '-GTCGCTCGAGTTATGTAACATCCTGGGGAGGCTCC-3' (IRAK2-2), (SEQ.I.D.NO:8) '-CGAATTCGACCATCAAATCCGGGAGCTGACTGC-3' (TRAF6-1 ), (SEQ.I.D.NO:9)

5'-GTCGCTCGAGCTATACCCCTGCATCAGTACTTCGTGGC-3' (TRAF6-2) (SEQ.I.D.NO:10)

PCR products were subcloned into pCMV-FLAG vector as EcoRI- Xhol fragments (pCMV-FLAG-IRAK2 and pCMV-FLAG-TRAF6-dominant negative respectively).

Transfections and Biochemical assay

5x105 HEK293 cells were transfected in duplicate in 6 well plates by a standard calcium-phosphate technique (Life Technology Ca-phosphate transfection system) using 2.5μg of total DNA. All transfections contain 1 μg of pNF-κB luc and 0.2μg of pRSV-b-galoctosidase (to normalise transfection efficiency). 0.5μg of pCMV-IRAK and pCMV-FLAG-IRAK3 expression plasmids were utilised, the concentration of either pCMV-FLAG-TRAF6- dominant negative or pCMV-FLAG-IRAK3-death domain was 1.3μg. DNA concentration was kept constant by supplementation of pcDNA- amp empty vector. 24h post transfection cells were collected and lysed in 100μl of lysis buffer (1 % Triton X-100, 25mM glycylglycine ph7.8, 15mM MgSO4 , 4mM EGTA, 1 mM DTT). Cell debris was collected by centrifugation at 12,000 rpm in Eppendorf centrifuge. Aliquots of cell lysate, 10μl, were mixed with 100μl of luciferase assay reagent (Promega), luciferase activity was determined using a luminescence counter (Wallac). β-Galacotisidase was assayed on 5μl of cell extracts in 100μl of b-galactosidase buffer (60 mM Na2HPO47H2O, 40mM NaH2PO4H2O, 2mM MgCI2, 2.7μl/ml b-Mercaptoethanol, 1 .33mg/ml o- nitrophenyi-β-D-Galactopyranoside). Samples were incubated at room temperature until a yellow colour developed and absorbance was read at 405 nm.

Relative luciferase activity was defined as the ratio between luciferase and β- Galactosidase activities obtained in each set of transfections.

HEK293 cells became responsive to IL-1 b stimulation (20pM) by co- transfection with human IL-1 receptor type l (1 μg of pCMV-lL-1 RI). The amount of total DNA used was 3.5μg, (1 μg of pNF-κB luc, 0.2μg of pRSV-b- galoctosidase, 1 .3μg of pCMV-FLAG-IRAK3DD). Cell stimulation was carried out for 8 hrs, 24hrs post transfection. Cell extracts were analysed as described above.

Screening for modulators of IRAK3

An IL-1 responsive cell line, such as HeLa or fibroblasts, is incubated in the presence of a candidate agent for 1 hr at 37C. These cells may then be stimulated with IL-1 for 1 hr at 37C before harvesting in ice-cold phosphate saline buffer (PBS) and cells pelleted at 1 ,500 g in a refrigerated centrifuge. Cells may be resuspended in lysis buffer containing: 40mM N-2- hydroxyethylpiperazine-N'-2-ethaneusulfonic acid (HEPES pH 7.8), 200mM NaCI, 3mM dithiothreitol (DTT), 30 mM β -glycerolphosphate, 5 mM p- nitrophenyl phosphate, 1 mM EDTA, 0.4% of NP-40, 1 mM Na-orthovanadate, 10% glycerol and protease inhibitors (Boehringer). Cells debris may be pelleted at 12,000g and supernatant aliquots used in a typical kinase assay, i.e. 20mM Hepes pH7.2, 50mM NaCI, 20mM MgCI2, 20 mM MnCI2, 30 mM β - glycerolphosphate, 2mM p-nitrophenyl phosphate, 0.1 mM Na-orthovanadate, 1 mM EDTA, 1 mM DTT, [γ -32P]ATP (10μCi), 10μM of cold ATP, in presence of 1 μg of IRAK3 polypeptide (or fragment thereof) suitably tagged (through e,g. either antibody epitope or biotinylated, etc) to allow its isolation and analysis of its phosphorylation state . Reactions may be incubated at 30° C for 30 min. Incorporation of 32P into IRAK3 polypeptide may be measured in a β-counter after specific capture of tagged peptide (through e.g. either anti-epitope or streptavidin coated plate). An effective candidate agent would be expected to inhibit the incorporation of 32P into the above polypeptide.

Bibliography

1 . Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87:2095.

2. Rosenwasser, L. J. 1998. BIOLOGIC ACTIVITIES OF IL-1 AND ITS ROLE IN HUMAN DISEASE. Journal of Allergy & Clinical Immunology 102:344.

3. Shelton, C. A. and S. A. Wasserman. 1993. Pelle encodes a protein kinase required to establish dorsoventral polarity in the Drosophila embryo. Cell 72:515.

4. Lemaitre, B., E. Nicolas, L. Michaut, J. M. Reichhart, and HoffmannJA.

1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973.

5. Martin, M. U. and W. Falk. 1997. The interleukin-1 receptor complex and interleukin-1 signal transduction. European Cytokine Network 8:5.

6. Oneill, L. A. J., C. Greene, Nf-kappa-b, Toll, Irak, Trafθ, N protei, and P38 map kinase. 1998. SIGNAL TRANSDUCTION PATHWAYS ACTIVATED BY THE IL-1 RECEPTOR FAMILY - ANCIENT SIGNALING MACHINERY IN MAMMALS, INSECTS, AND PLANTS. Journal of Leukocyte Biology 63:650. 7. Greenfeder, S. A., P. Nunes, L. Kwee, M. Labow, and R. A. J. Chizzonite,G. 1995. Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. Journal Of Biological Chemistry 270:13757.

8. Huang, J. N., X. Gao, S. Li, and Z. D. Cao. 1997. RECRUITMENT OF IRAK TO THE INTERLEUKIN 1 RECEPTOR COMPLEX REQUIRES INTERLEUKIN 1 RECEPTOR ACCESSORY PROTEIN. Proceedings Of The National Academy Of Sciences Of The United States Of America 94:12829.

9. Volpe, F., J. Clatworthy, A. Kaptein, B. Maschera, A. M. Griffin, K. Ray.

1997. THE 1L1 RECEPTOR ACCESSORY PROTEIN IS RESPONSIBLE FOR THE RECRUITMENT OF THE INTERLEUKIN-1 RECEPTOR ASSOCIATED KINASE TO THE IL1/IL1 RECEPTOR I COMPLEX. Febs Letters 419:41.

10. Muzio, M., J. Ni, P. Feng, and V. M. Dixit. 1997. IRAK (PELLE) FAMILY MEMBER IRAK-2 AND MYD88 AS PROXIMAL MEDIATORS OF IL-1 SIGNALING. Science 278:1612.

1 1. Wesche, H., W. J. Henzel, W. Shillinglaw, S. Li, and Z. D. Cao. 1997. MYD88 - AN ADAPTER THAT RECRUITS IRAK TO THE IL-1 RECEPTOR COMPLEX. Immunity 7:837.

12. Burns, K., F. Martinon, C. Esslinger, H. Pahl, P. Schneider, J. L. Bodmer, F. Dimarco, L. French, and J. Tschopp. 1998. MYD88, AN

ADAPTER PROTEIN INVOLVED IN INTERLEUKIN-1 SIGNALING. Journal Of Biological Chemistry 273:12203.

13. Martin, M., G. F. Bol, A. eriksson, K. Resh, and R. Brigelius Flohe. 1994. Interleukin-1 -induced activation of a protein kinase co-precipitating with the type I interiukin-1 receptor in T cells. European Journal Immunology 24:1566. 14. Crostoπ, G. E., Z. Cao, and D. V. Goeddel. 1995. NF-kappa B activation by interleukin-1 (IL-1 ) requires an IL-1 receptor- associated protein kinase activity. J. Biol. Chem. 270:16514.

15. Cao, Z., W. J. Henzel, and X. Gao. 1996. IRAK: a kinase associated with the interleukin-1 receptor. Science 271 :1 128.

16. Muzio, M., G. Natoli, and S. Saccani. 1998. THE HUMAN TOLL SIGNALING PATHWAY - DIVERGENCE OF NUCLEAR FACTOR KAPPA-B

AND JNK/SAPK ACTIVATION UPSTREAM OF TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTOR 6 (TRAF6). Journal Of Experimental Medicine 187:2097.

17. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, and D. V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature 383:443.

18. Malinin, N. L., M. P. Boldin, A. V. Kovalenko, and D. Wallach. 1997. MAP3K-RELATED KINASE INVOLVED IN NF-KAPPA-B INDUCTION BY TNF, CD95 AND IL-1 . Nature 385:540.

19. Song, H. Y., C. H. Regnier, C. J. Kirschning, D. V. Goeddel, and M. Rothe. 1997. Tumor necrosis factor (tnf)-mediated kinase cascades - bifurcation of nuclear factor-kappa-b and c-jun n-terminal kinase (jnk sapk) pathways at tnf receptor-associated factor 21. Proceedings Of The National Academy Of Sciences Of The United States Of America 94:9792.

20. Didonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, and M. Karin.

1997. A CYTOKINE-RESPONSIVE l-KAPPA-B KINASE THAT ACTIVATES THE TRANSCRIPTION FACTOR NF-KAPPA-B. Nature 388:548. 21. Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, and M. Karin.

1997. THE l-KAPPA-B KINASE COMPLEX (IKK) CONTAINS TWO KINASE SUBUNITS, IKK-ALPHA AND IKK-BETA, NECESSARY FOR l-KAPPA-B PHOSPHORYLATION AND NF-KAPPA-B ACTIVATION. Cell 91 :243.

22. Rothwar , D. M., E. Zandi, G. Natoli, and M. Karin. 1998. IKK-GAMMA IS AN ESSENTIAL REGULATORY SUBUNIT OF THE l-KAPPA-B KINASE COMPLEX. Nature 395:297.

23. Yamin, T. T. and D. K. Miller. 1997. The interleukin-1 receptor- associated kinase is degraded by proteasomes following its phosphorylation. Journal Of Biological Chemistry 272:21540. 24. Arend W.P. et al. 1998. Interleukin - 1 Receptor Antagonist: Role in Biology. Annu.Rev.lmmunol. 16:27-55. 25. Verma IM. Stevenson JK. Schwarz EM. Van Antwerp D. Miyamoto S. 1995. Rel/NF-kappa B/l kappa B family: intimate tales of association and dissociation. Genes & Development. 9(22):2723-35.

Claims

Claims
1. An isolated mammalian protein comprising the ammo acid sequence as set forth in SEQ.I.D.NO:1 , SEQ.I.D.NO:3 or a variant thereof.
2. The protein of claim 1 wherein the variant is a fragment.
3. The protein of claim 2 wherein the fragment is the death domain, kinase domain or carboxyl terminal domain.
4 The protein of claim 1 wherein the variant is a derivative which has a substitution, deletion or insertion of one or more ammo acids.
5. The protein of claim 4 wherein the derivative is a non-naturally occunng protein in which one or more ammo acids of SEQ.I.D.NO.1 , SEQ.l.D.NO.3 or fragment thereof has been subsituted, deleted, rearranged or modified yet biological activity or function or its effectiveness in a screen is substantially retained.
6. The protein of claim 4 wherein the derivative is a non-naturally occunng protein in which one or more ammo acids have been added or inserted into the protein having the ammo acid sequence as set forth in SEQ.I.D.NO.1 , SEQ.l.D.NO.3 or fragment thereof.
7 A protein according to any one of the preceeding claims having at least 70% identity with the protein having a primary ammo acid sequence as set forth in SEQ I.D.NO.1 or SEQ.l.D.NO.3.
8 A protein according to claim 7 having at least 80% identity with the protein having a primary ammo acid sequence as set forth in SEQ. I. D. NO 1 or SEQ I.D NO:3.
9. A protein according to claim 1 which is encoded by a polynucleotide comprising a sequence as set forth in SEQ.I.D.NO.2.
10 A polynucleotide comprising the sequence as set forth in SEQ.I.D.NO.2.
1 1. A polynucleotide according to claim 10 further comprising a start codon.
12. A polynucleotide which is equivalent to the polynucleotide as set forth in SEQ.I.D.NO:2 as a result of the redundancy of the genetic code.
13. A vector comprising a polynucleotide according to any one of claims 9 to 12.
14. A vector according to claim 13 further comprising regulatory elements.
15. A host cell comprising the vector of claim 13 or 14.
16. An anti-sense strand of the sequence of SEQ.I.D.NO:2 or fragment thereof.
17. Antibody, or fragment thereof, capable of binding to the protein as claimed in any one of claims 1 to 9.
18. A modulator of the protein as claimed in any one of claims 1 to 9.
19. A modulator according to claim 18 wherein the modulator is an antagonist.
20. A modulator according to claim 18 or 19 which disrupts or perturbs the function or activity of one or more of the domains of the protein as set forth in SEQ.I.D.NO:1 or SEQ.I.D.NO-3.
21 . A modulator according to claim 18, 19 or 20 which inhibits phosphorylation of a protein as claimed in any one of claims 1 to 9.
22. A modulator according to claim 21 which inhibits phosphorylation of the protein as set forth in SEQ.I.D.NO:1 or SEQ.I.D.NO:3 at position 69.
23. A medicament comprising a therapeutically effective amount of a modulator according to any one of claims 18 to 22.
24. A medicament according to claim 23 further comprising other therapeutic agents.
25. A method of treating a disease or condition affected by IL-1 family activity comprising the step of administrating a therapeutically effective amount of the modulator as claimed in any one of claims 1 to 9.
26. Use of the modulator of claims 18 to 22 in the manufacture of a medicament for the treatment of dieases or conditions affected by IL-1 family activity.
27. A method of screening a candidate compound for its ability to antagonise the biological function or activity of a protein as claimed in any one of claims 1 to 9 comprising the steps of;
(a) stimulating a IL-1 responsive cell with a IL-1 agonist in the presence of the candidate compound (optionally which binds to the protein as set forth in SEQ.I.D.NO.1 );
(b) measuring the degree of stimulation;
(c) optionally comparing the degree of stimulation with control.
28. The method of claim 27 wherein the step of measuring involves determining the amount of 32P incorporation into IRAK3.
PCT/EP2000/005290 1999-06-08 2000-06-08 Interleukin-1-receptor associated kinase-3 (irak3) WO2000075310A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
GB9913180.7 1999-06-08
GBGB9913180.7A GB9913180D0 (en) 1999-06-08 1999-06-08 Interleukin-1 receptor associated kinases

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
AU54039/00A AU5403900A (en) 1999-06-08 2000-06-08 Interleukin-1-receptor associated kinase-3 (irak3)

Publications (2)

Publication Number Publication Date
WO2000075310A1 true WO2000075310A1 (en) 2000-12-14
WO2000075310A8 WO2000075310A8 (en) 2001-02-08

Family

ID=10854859

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/005290 WO2000075310A1 (en) 1999-06-08 2000-06-08 Interleukin-1-receptor associated kinase-3 (irak3)

Country Status (3)

Country Link
AU (1) AU5403900A (en)
GB (1) GB9913180D0 (en)
WO (1) WO2000075310A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7132438B2 (en) 2001-10-09 2006-11-07 Amgen Inc. Benzimidazole derivatives

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000690A1 (en) * 1995-06-23 1997-01-09 Tularik, Inc. Interleukin-1 receptor-associated protein kinase and assays
WO1999027112A1 (en) * 1997-11-26 1999-06-03 Human Genome Sciences, Inc. Human irak-2, a human interleukin-1 receptor associated kinase-2
WO2000009678A1 (en) * 1998-08-17 2000-02-24 Tularik Inc. Irak3 polypeptides, polynucleotides and methods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000690A1 (en) * 1995-06-23 1997-01-09 Tularik, Inc. Interleukin-1 receptor-associated protein kinase and assays
WO1999027112A1 (en) * 1997-11-26 1999-06-03 Human Genome Sciences, Inc. Human irak-2, a human interleukin-1 receptor associated kinase-2
WO2000009678A1 (en) * 1998-08-17 2000-02-24 Tularik Inc. Irak3 polypeptides, polynucleotides and methods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL - EMEST_HUM2 30 March 1998 (1998-03-30), STRAUSBERG, R.: "am11f01.s1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone IMAGE:1466521 3', mRNA sequence.", XP002147674 *
WESCHE HOLGER ET AL: "IRAK-M is a novel member of the pelle/interleukin-1 receptor-associated kinase (IRAK) family.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 27, 2 July 1999 (1999-07-02), pages 19403 - 19410, XP002147673, ISSN: 0021-9258 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7132438B2 (en) 2001-10-09 2006-11-07 Amgen Inc. Benzimidazole derivatives
US7635774B2 (en) 2001-10-09 2009-12-22 Amgen Inc. Benzimidazole derivatives

Also Published As

Publication number Publication date
WO2000075310A8 (en) 2001-02-08
GB9913180D0 (en) 1999-08-04
AU5403900A (en) 2000-12-28

Similar Documents

Publication Publication Date Title
ES2284199T5 (en) Receiver 4 containing death domain (DR4: death receptor 4) superfamily member and TNF receptor binding trail (apo-2l).
US6288032B1 (en) Osteoprotegerin
Beer et al. Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression
CN1107072C (en) Programing cell death induced molecule II
KR100507431B1 (en) Tumor Necorsis Factor-γ
CA2395539C (en) Soluble interleukin-20 receptor
Maruyama et al. A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells
EP0897390B1 (en) Human tumor necrosis factor delta and epsilon
US6664227B1 (en) Treatment of fibrosis by antagonism of IL-13 and IL-13 receptor chains
DE60121808T2 (en) Use of a polypeptide comprising the extracellular domain of IL-20RA and IL-20RB, the treatment of inflammation
US20030148445A1 (en) TALL-1 nucleic acid molecules, proteins, receptors and methods of use thereof
Madry et al. The characterization of murine BCMA gene defines it as a new member of the tumor necrosis factor receptor superfamily.
EP0853668B1 (en) Regulated genes and uses thereof
Goujon et al. Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction.
US20020127651A1 (en) Human 4-1BB receptor splicing variant
AU760224B2 (en) Isolated nucleic acid molecules which encode T cell inducible factors (TIFs), the proteins encoded, and uses thereof
DE69531892T2 (en) Fibroblast growth factor 10
EP1022286A1 (en) POLYPEPTIDE, cDNA ENCODING THE POLYPEPTIDE, AND USE OF THE BOTH
WO1997033899A1 (en) Apoptosis inducing molecule i
US5552303A (en) DNA encoding epithelium-derived T-cell factor
US5942417A (en) CD44-like protein and nucleic acids
JP2008081503A (en) Death domain-containing receptor 5
US6703360B2 (en) Compositions and methods related to canine IgG and canine IL-13 receptors
US5780291A (en) Wnt-x growth factor polypeptide, DNA encoding same, and Wnt-x antibody
EP0620739A1 (en) Method of treating tnf-dependent inflammation using tumor necrosis factor antagonists

Legal Events

Date Code Title Description
AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ 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

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ 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 MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: C1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ 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 MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

WR Later publication of a revised version of an international search report
AL Designated countries for regional patents

Kind code of ref document: C1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ 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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP