Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-08-11
2001-04-03
Spector, Lorraine (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S194000, C436S501000
Reexamination Certificate
active
06211337
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of this invention is a novel human kinase involved in tumor necrosis factor signal transduction and its use in drug screening.
2. Background
Tumor necrosis factor (TNF) is an important cytokine involved in the signaling of a number of cellular responses including cytotoxicity, anti-viral activity, immun.-regulatory activities and the transcriptional regulation of a number of genes. The TNF receptors (TNF-R1 and TNF-R2) are members of the larger TNF receptor superfamily which also includes the Fas antigen, CD27, CD30, CD40, and the low affinity nerve growth factor receptor. Members of this family have been shown to participate in a variety of biological properties, including programmed cell death, antiviral activity and activation of the transcription factor NF-&kgr;B in a wide variety of cell types.
Accordingly, it is desired to identify agents which specifically modulate transduction of TNF receptor family signaling. Unfortunately, the components of the signaling pathway remain largely unknown; hence, the reagents necessary for the development of high-throughput screening assays for such therapeutics are unavailable. Elucidation of TNF receptor family signal transduction pathways leading to NF-&kgr;B activation would provide valuable insight into mechanisms to alleviate inflammation. In particular, components of this pathway would provide valuable targets for automated, cost-effective, high throughput drug screening and hence would have immediate application in a broad range of domestic and international pharmaceutical and biotechnology drug development programs.
Relevant Literature
Stanger et al. (1995) Cell 81, 513-523 report the existence of a Receptor Interacting Protein (RIP) and its functional expression. VanArsdale and Ware (1994) J Immunology 153:3043-3050 describe proteins associated with TNF-R1. The cloning and amino acid sequencing of TNF-R1 is disclosed in Schall et al (1990) Cell 61, 361 and Loetscher et al (1990) Cell 61, 351; the identification of a “death domain” in TNF-R1 is disclosed in Tartaglia et al. (1993) Cell 74:845-853. The cloning and amino acid sequence of a TNF-R associated death domain protein (TRADD) is described by Hsu et al. (1995) Cell 81, 495-504. The cloning and amino acid sequence of the Fas antigen is disclosed in Itoh et al (1991) Cell 66, 233-243. For a recent review, see Smith et al. (1994) Cell 76:959-962 and Vandenabelle et al. (1995) Trends Cell Biol. 5, 392-399.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to a human Receptor Interacting Protein (hRIP). The compositions include nucleic acids which encode hRIP, hRIP kinase domains, and recombinant proteins made from these nucleic acids. The invention also provides methods for screening chemical libraries for lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated hRIP activity or hRIP-dependent signal transduction. In one embodiment, the methods involve incubating a mixture of hRIP, a natural intracellular hRIP substrate or binding target and a candidate pharmacological agent and determining if the presence of the agent modulates the ability of hRIP to selectively phosphorylate the substrate or bind the binding target. Specific agents provide lead compounds for pharmacological agents capable of disrupting hRIP function.
DETAILED DESCRIPTION OF THE INVENTION
A human RIP-encoding nucleic acid sequence is set out in SEQ ID NO: 1. A human RIP kinase domain-encoding nucleic acid sequence is set out in SEQ ID NO: 1, nucleotides 1-900. A human RIP amino acid sequence is set out in SEQ ID NO: 2; and a hRIP kinase domain sequence is set out in SEQ ID NO:2, residues 1-300.
Natural nucleic acids encoding hRIP are readily isolated from cDNA libraries with PCR primers and hybridization probes containing portions of the nucleic acid sequence of SEQ ID NO:1. For example, we used low stringency hybridization at 42° C. (hybridization buffer: 20% formamide, 10% Denhardt, 0.5% SDS, 5×SSPE; with membrane washes at room temperature with 5×SSPE/0.5% SDS) with a 120 base oligonucleotide probe (SEQ ID NO: 1, nucleotides 1728-1847) to isolate a native human RIP cDNA from a library prepared from human umbilical vein endothelial cells. In addition, synthetic hRIP-encoding nucleic acids may be generated by automated synthesis.
The subject nucleic acids are recombinant, meaning they comprise a sequence joined to a nucleotide other than that to which sequence is naturally joined and isolated from a natural environment. The nucleic acids may be part of hRIP-expression vectors and may be incorporated into cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for disease associated with expression of a hRIP), etc. These nucleic acids find a wide variety of applications including use as templates for transcription, hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of hRIP genes and gene transcripts, in detecting or amplifying nucleic acids encoding additional hRIP homologs and structural analogs, and in gene therapy applications.
In a particular embodiment, the invention provides RIP-Thr
514
polypeptides, RIP-Thr
514
polypeptide-encoding nucleic acids/polynucleotides, and RIP-Thr
514
polypeptide-based methods (below), which RIP-Thr
514
polypeptides comprise at least 8, preferably at least 10, more preferably at least 12, more preferably at least 16, most preferably at least 24 consecutive amino acid residues of the amino acid sequence set forth as SEQ ID NO:2, which consecutive amino acid residues comprise the amino acid residue 514 (Thr) of SEQ ID NO:2. Exemplary RIP-Thr
514
polypeptides having RIP-Thr
514
binding specificity and immunologically distinguishable from RIP-Ser
514
are shown in Table I.
TABLE I. Exemplary RIP-Thr
514
Polypeptides Having RIP-Thr
514
Binding Specificity
&agr;&Dgr;1 (SEQ ID NO:2, residues 509-518)
&agr;&Dgr;2 (SEQ ID NO:2, residues 514-521)
&agr;&Dgr;3 (SEQ ID NO:2, residues 506-514)
&agr;&Dgr;4 (SEQ ID NO:2, residues 504-524)
&agr;&Dgr;5 (SEQ ID NO:2, residues 498-514)
&agr;&Dgr;6 (SEQ ID NO:2, residues 514-534)
&agr;&Dgr;7 (SEQ ID NO:2, residues 513-520)
&agr;&Dgr;8 (SEQ ID NO:2, residues 508-515)
&agr;&Dgr;9 (SEQ ID NO:2, residues 512-522)
&agr;&Dgr;10 (SEQ ID NO:2, residues 423-514)
&agr;&Dgr;11 (SEQ ID NO:2, residues 423-543)
&agr;&Dgr;12 (SEQ ID NO:2, residues 423-579)
&agr;&Dgr;13 (SEQ ID NO:2, residues 423-633)
&agr;&Dgr;14 (SEQ ID NO:2, residues 423-671)
&agr;&Dgr;15 (SEQ ID NO:2, residues 514-543)
&agr;&Dgr;16 (SEQ ID NO:2, residues 514-579)
&agr;&Dgr;17 (SEQ ID NO:2, residues 514-633)
&agr;&Dgr;18 (SEQ ID NO:2, residues 514-671)
In a particular embodiment, the invention provides RIP-ACA
1540-1542
polynucleotides, comprising at least 18, 24, 36, 48, 72, 148, 356 or 728 consecutive nucleotides of the nucleotide sequence set forth as SEQ ID NO:1, which consecutive polynucleotides comprise the polynucleotides 1540-1542 (ACA) of SEQ ID NO:1. Exemplary RIP-ACA
1540-1542
polynucleotides and allele specific oligonucleotide probes having RIP-ACA
1540-1542
binding specificity and distinguishable by hybridization assays from RIP-TCT
1540-1542
are shown in Table II.
TABLE II. Exemplary RIP-ACA
1540-1542
Polynucleotides Having RIP-ACA
1540-1542
Binding Specificity
&agr;&Dgr;1 (SEQ ID NO:1, nucleotides 1540-1557)
&agr;&Dgr;2 (SEQ ID NO:1, nucleotides 1540-1563)
&agr;&Dgr;3 (SEQ ID NO:1, nucleotides 1540-1675)
&agr;&Dgr;4 (SEQ ID NO:1, nucleotides 1540-1699)
&agr;&Dgr;5 (SEQ ID NO:1, nucleotides 1525-1542)
&agr;&Dgr;6 (SEQ ID NO:1, nucleotides 1519-1542)
&agr;&Dgr;7 (SEQ ID NO:1, nucleotides 1507-1542)
&agr;&Dgr;8 (SEQ ID NO:1, nucleotides 1483-1542)
&agr;&Dgr;9 (SEQ ID NO:1, nucleotides 1537-1545)
&agr;&Dgr;10 (SEQ ID NO:1, nucleotides 1534-1548)
&agr;&Dgr;11 (SEQ ID NO:1, nucleotides 1528-1554)
&agr;&Dgr;12 (SEQ ID NO:1, nucleotides 1516-1566)
&agr;&Dgr;13 (SEQ ID NO:1, nucleotides 1504-1554)
&agr;&Dgr;14 (SEQ ID NO:1, nucleot
Baichwal Vijay R.
Goeddel David V.
Hsu Hailing
Huang Jianing
Lazar-Wesley Eliane
Osman Richard Aron
Spector Lorraine
Tularik Inc.
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