Novel interleukin-1 receptor intracellular ligand proteins and inhibitors of ligand binding

a technology of interleukin-1 receptor and intracellular ligand, which is applied in the field of antiinflammatory substances, can solve the problems of ligands which bind to the il-1-r intracellular domain that have yet to be identified, and achieve the effect of preventing or ameliorating an inflammatory condition

Inactive Publication Date: 2005-08-25
GENETICS INST INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ligands which bind to the IL-1-R intracellular domain have yet to be identified.

Method used

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  • Novel interleukin-1 receptor intracellular ligand proteins and inhibitors of ligand binding
  • Novel interleukin-1 receptor intracellular ligand proteins and inhibitors of ligand binding

Examples

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example 1

Cloning of IL-1-R Intracellular Ligand Protein Encoding Polynucleotide

[0110] A yeast genetic selection method, the “interaction trap” [Gyuris et al, Cell 75:791-803, 1993, which is incorporated herein by reference], was used to screen W138 and HeLa cell cDNA libraries (preparation see below) for proteins that interact with IL-1-R-1c, the cytoplasmic portion (intracellular domain) of the interleukin-1 receptor p80, or type I. The IL-1-R-1c DNA, encoding amino acids 340 to 552 of the type I IL-1 receptor, was obtained via the polymerase chain reaction (PCR) of a human W138 cell cDNA library. This IL-1-R-1c DNA was then cloned into pEG202 by an EcoRI site, generating the bait plasmid, pEG202-IL-1-R-1c. This plasmid contains the HIS3 selectable marker, and expression of the bait, the LexA-IL-1-R-1c fusion protein, is from the strong constitutive ADH1 promoter. To create the reporter strain carrying the bait protein, yeast strain EGY48, containing the reporter sequence LexAop-Leu2 in pl...

example 2

Expression of the IL-1-R Intracellular Ligand Protein

[0120] cDNAs encoding IL-1-R intracellular ligand proteins were released from the pJG4-5 vector with the appropriate restriction enzymes. For example. EcoRI and XhoI were used to release cDNA from the relevant clone. Where the restriction sites were also present in the internal sequence of the cDNA. PCR was performed to obtain the cDNA. These cDNAs were then cloned into various expression vectors. These included pGEX (Pharmacia) or pMAL (New England Biolabs) for expression as a GST (Glutathione-S-transferase) or MBP (maltose binding protein) fusion protein in E. coli, a pED-based vector for mammalian expression, and pVL or pBlueBacHis (Invitrogen) for baculovirus / insect expression. For the immunodetection of IL-1-R intracellular ligand expression in mammalian cells. an epitope sequence, “Flag,” was inserted into the translational start site of the pED vector, generating the pED-Flag vector. cDNAs were then inserted into the pED-F...

example 3

Assays of IL-1-R Intracellular Domain Binding

[0122] Two different methods were used to assay for IL-1-R intracellular ligand protein activity. The first assay measures binding in the yeast strain in “interaction trap,” the system used here to screen for IL-1-R-1c interacting proteins. In this system, the expression of reporter genes from both LexAop-Leu2 and LexAop-LacZ relies on the interaction between the bait protein, in this case IL-1-R-1c, and the prey, the IL-1-R intracellular ligand. Thus, one can measure the strength of the interaction by the level of Leu2 or LacZ expression. The most simple method is to measure the activity of the LacZ encoded protein, β-galactosidase. This activity can be judged by the degree of blueness on the X-Gal containing medium or filter. For the quantitative measurement of β-galactosidase activity, standard assays can be found in “Methods in Yeast Genetics” Cold Spring Harbor, New York, 1990 (by Rose, M. D., Winston, F., and Hieter, P.).

[0123] Th...

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Abstract

Novel IL-1-R intracellular ligand proteins are disclosed. Polynucleotides encoding the IL-1-R intracellular ligand protein are also disclosed, along with vectors, host cells, and methods of making the IL-1-R intracellular ligand protein. Pharmaceutical compositions containing the IL-1-R intracellular ligand protein, methods of treating inflammatory conditions, and methods of inhibiting IL-1-R intracellular domain binding are also disclosed. Methods of identifying inhibitors of IL-1-R intracellular domain binding and inhibitors identified by such methods are also disclosed.

Description

BACKGROUND OF THE INVENTION [0001] The present invention relates to the field of anti-inflammatory substances and other substances which act by inhibiting binding to the intracellular domain of an interleukin-1 receptor (hereinafter “IL-1-R”), such as, for example, the p80, type I IL-1 receptor. More particularly, the present invention is directed to novel ligands which bind to the IL-1-R intracellular domain and to inhibition or modulation of signal transduction by this receptor. [0002] Interleukin-1-α and interleukin-1-β (herein collectively “IL-1”) are cytokines which produce a wide range of cellular activities. IL-1 causes an inflammatory response, which can be beneficial, such as in mounting an immune response to a pathogen, or when overexpressed can lead to other detrimental effects of inflammation. [0003] The cellular effects of IL-1 are initiated by the binding of IL-1 to its receptors (IL-1-Rs) on the surface of target cells. The isolation of polynucleotides encoding IL-1-R...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/47C12N15/12
CPCC07K14/47A61K38/00A61P29/00
Inventor LIN, LIH-LINGGRAHAM, JAMES
Owner GENETICS INST INC
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