Aggrecanase molecules

a technology of aggrecanase and aggrecanase, which is applied in the field of nucleotide sequences encoding novel aggrecanase molecules, can solve the problems of cartilage biomechanical characteristics deficiencies, severe osteoarthritis reducing quality of life, etc., and achieves the effect of preventing aggrecan cleavage, inhibiting or antagonizing the activity of aggrecanase, and reducing the activity of aggrecan

Inactive Publication Date: 2007-06-07
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The invention also provides antibodies that bind to isolated aggrecanase proteins of the invention. In one embodiment, such an antibody reduces, inhibits or antagonizes aggrecanase activity. The invention further provides methods for developing and identifying inhibitors of aggrecanase activity comprising the use of aggrecanase protein chosen from SEQ ID NO: 2 or a fragment or a variant thereof. In one embodiment, inhibitors of aggrecanase activity prevent cleavage of aggrecan.

Problems solved by technology

Osteoarthritis can severely reduce quality of life due to degradation of articular cartilage and the resulting chronic pain.
The large, sugar-containing portion of aggrecan is thereby lost from the extra-cellular matrix, resulting in deficiencies in the biomechanical characteristics of the cartilage.

Method used

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example 1

Isolation of DNA

[0103] Potential novel aggrecanase family members were identified using a database screening approach. Aggrecanase-1 (Science 284:1664-1666 (1999)) has at least six domains: signal, propeptide, catalytic domain, disintegrin, tsp (thrombospondin), and c-terminal. The catalytic domain contains a zinc binding signature region, TAAHELGHVKF (SEQ. ID NO: 6) and a “MET turn” which are responsible for protease activity. Substitutions within the zinc binding region in the number of the positions still allow protease activity, but the histidine (H) and glutamic acid (E) residues must be present. The thrombospondin domain of Aggrecanase-1 is also a critical domain for substrate recognition and cleavage. It is these two domains that determine our classification of a novel aggrecanase family member. The coding region of the aggrecanase-1 DNA sequence was used to query against the GeneBank ESTs focusing on human ESTs using TBLASTN. The resulting sequences were the starting point...

example 2

EST18 Tissue Expression

[0111] A Clontech human multiple tissue expression array MTE™ (Clontech Catalog #: 7776-1) was probed with a 533 base pair α-32P dCTP-labeled CDNA probe according to the manufacturer's guidelines. Probe labeling and hybridization were performed as follows: 5 μg of Al 18FS plasmid (described below) was digested with EcoRI enzyme in its optimal buffer according to the vendor's recommendations. The restriction digest was fractionated on a 1% agarose gel and a 533 base pair fragment encoding EST18 protein sequence including amino acid #1 (methionine) through amino acid #174 (asparagine) of SEQ ID NO: 2 was recovered from the agarose gel as outlined above. An α-32P dCTP-labeled probe was made utilizing Amersham Pharmacia's Ready-To-Go kit (Catalog #: 27-9240-01, Pharmacia, ). Briefly, 30 ng of heat-denatured DNA was incubated at 37° C.for 15 minutes with 50 μCi of α-32P dCTP and one labeling bead. Following the incubation, the reaction mix was applied to a pre-eq...

example 3

Expression of a Truncated form the Aggrecanase Protein

[0113] A truncated form of protein encoded by the EST18 nucleotide sequence was expressed as a fusion protein. One such truncated protein, A18FS, refers to the first 650 amino acids, from amino acid #1 (methionine) to amino acid #650 (phenylalanine) encoded by the EST18 nucleotide sequence. The expression construct was generated in two steps. First, the 5′ end of EST18 nucleotide sequence was modified to include the additional coding nucleotide sequence identified by 5′ RACE. Second, the construct had an open reading frame, such that it ended at the codon for phenylalanine. A Streptavidin-Tag sequence was added to aid in purification of the recombinant protein.

[0114] Modification of the 5′ end: The six synthetic oligonucleotides listed below were designed to anneal together to form a DNA sequence flanked by an EcoRI site on the 5′ end and a Sacl site on the 3′ end. The cloned EST18 sequence was digested with EcoRI and SacI enz...

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Abstract

Novel aggrecanase proteins and the nucleotide sequences encoding them as well as processes for producing them are disclosed. Methods of identifying and developing inhibitors of the aggrecanase enzymes and antibodies to the enzymes for treatment of conditions characterized by the degradation of aggrecan are also disclosed.

Description

RELATED APPLICATION [0001] This application relies on the benefit of priority of U.S. provisional patent application No. 60 / 353,680, filed on Jan. 31, 2002, the entire disclosure of which is incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to the discovery of nucleotide sequences encoding novel aggrecanase molecules, aggrecanase proteins and fragments thereof, and processes for producing them. The invention further relates to identification and development of inhibitors of and antibodies to the aggrecanase enzymes. These inhibitors and antibodies may be useful for the treatment of various aggrecanase-associated conditions including osteoarthritis. BACKGROUND OF THE INVENTION [0003] Aggrecan is a major extracellular component of articular cartilage. It is a proteoglycan responsible for providing cartilage with its mechanical properties of compressibility and elasticity. The loss of aggrecan has been implicated in the degradation of articular carti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/53G06F19/00C07H21/04C12N9/64C07K16/40C12N15/09A61K39/395A61K45/00A61P1/00A61P1/02A61P1/04A61P7/00A61P9/00A61P11/00A61P11/06A61P13/00A61P17/02A61P19/00A61P19/02A61P19/10A61P25/00A61P25/02A61P25/06A61P25/14A61P25/16A61P25/24A61P25/28A61P27/02A61P29/00A61P35/00A61P37/02A61P37/06A61P37/08A61P43/00C07K16/18C12N1/15C12N1/19C12N1/21C12N5/10C12N5/22C12P21/02C12Q1/37
CPCA61K2039/505C12N9/6421A61P1/00A61P1/02A61P1/04A61P7/00A61P9/00A61P11/00A61P11/06A61P13/00A61P17/02A61P19/00A61P19/02A61P19/10A61P25/00A61P25/02A61P25/06A61P25/14A61P25/16A61P25/24A61P25/28A61P27/02A61P29/00A61P35/00A61P37/02A61P37/06A61P37/08A61P43/00C12N9/64C12N15/52
Inventor CORCORAN, CHRISTOPHER J.AGOSTINO, MICHAEL J.LAVALLIE, EDWARD R.FLANNERY, CARL R.ZENG, WEILANCOLLINS-RACIE, LISA A.FREEMAN, BETHANY A.
Owner WYETH LLC
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