Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-09-09
2002-05-28
Achutamurthy, Ponnathapu (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C435S069100, C435S252300, C435S320100
Reexamination Certificate
active
06395889
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to newly identified polynucleotides having homology to various protease families. The invention also relates to protease polypeptides. The invention further relates to methods using the protease polypeptides and polynucleotides as a target for diagnosis and treatment in protease-mediated and related disorders. The invention further relates to drug-screening methods using the protease polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the protease polypeptides and polynucleotides. The invention further relates to procedures for producing the protease polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
Proteases are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown protease nucleic acids and polypeptides. The present invention advances the state of the art by providing previously unidentified human protease sequences.
SUMMARY OF THE INVENTION
It is an object of the invention to identify novel proteases.
It is a further object of the invention to provide novel protease polypeptides that are useful as reagents or targets in protease assays applicable to treatment and diagnosis of protease-mediated disorders.
It is a further object of the invention to provide polynucleotides corresponding to the novel protease polypeptides that are useful as targets and reagents in protease assays applicable to treatment and diagnosis of protease-mediated disorders and useful for producing novel protease polypeptides by recombinant methods.
A specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of the novel proteases.
A further specific object of the invention is to provide compounds that modulate expression of the proteases for treatment and diagnosis of protease- related disorders.
The present invention is based on the identification of novel nucleic acid molecules that are homologous to protease sequences.
Thus, in one aspect, the invention provides an isolated nucleic acid molecule that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-33 and the complements of SEQ ID NOS: 1-33.
In another aspect, the invention provides isolated proteins and polypeptides encoded by nucleic acid molecules of the invention.
In another embodiment, the invention provides an isolated nucleic acid molecule that comprises a nucleotide sequence that is at least about 60% identical, preferably at least about 80% identical, preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical, and most preferably about 98% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-33 and the complements of SEQ ID NOS: 1-33.
The invention also provides isolated variant polypeptides.
The invention also provides an isolated fragment or portion of any of SEQ ID NOS: 1-33 and the complement of SEQ ID NOS: 1-33. In preferred embodiments, the fragment is useful as a probe or primer, and/or is at least 15, more preferably at least 18, even more preferably 20-25, 30, 50, 100, 200 or more nucleotides in length.
The invention also provides isolated fragments of the polypeptides.
In another embodiment, the invention provides an isolated nucleic acid molecule that hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-33 and the complements of SEQ ID NOS: 1-33.
The invention further provides nucleic acid constructs comprising the nucleic acid molecules described above. In a preferred embodiment, the nucleic acid molecules of the invention are operatively linked to a regulatory sequence.
The invention also provides vectors and host cells for expressing the protease nucleic acid molecules and polypeptides and particularly recombinant vectors and host cells.
The invention also provides methods of making the vectors and host cells and methods for using them to produce the protease nucleic acid molecules and polypeptides.
The invention also provides antibodies or antigen-binding fragments thereof that selectively bind the protease polypeptides and fragments.
The invention also provides methods of screening for compounds that modulate expression or activity of the protease polypeptides or nucleic acid (RNA or DNA).
The invention also provides a process for modulating protease polypeptide or nucleic acid expression or activity, especially using the screened compounds. Modulation may be used to treat conditions related to aberrant activity or expression of the protease polypeptides or nucleic acids.
The invention also provides assays for determining the presence or absence of and level of the protease polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
The invention also provides assays for determining the presence of a mutation in the protease polypeptides or nucleic acid molecules, including for disease diagnosis.
In still a further embodiment, the invention provides a computer readable means containing the nucleotide and/or amino acid sequences of the nucleic acids and polypeptides of the invention, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The following text describes classes of proteins with which the sequences described herein have been compared with respect to homology.
Eukaryotic and Viral Aspartyl Active Sites
Aspartyl proteases, also known as acid proteases, (EC 3.4.23.-), are a widely distributed family of proteolytic enzymes (Foltman (1981)
Essays Biochem
17:52-84; Davis (1990)
Annu. Rev. Biophys. Chem
. 19: 189-215; Rao et al. (1991)
Biochemistry
30: 4663-4671) that exist in vertebrates, fungi, plants, retroviruses and some plant viruses. Aspartate proteases of eukaryotes are monomeric enzymes which consist of two domains. Each domain contains an active site centered on a catalytic aspartyl residue. The two domains most probably evolved from the duplication of an ancestral gene encoding a primordial domain. Currently known eukaryotic aspartyl proteases include, but are not limited to:
Vertebrate gastric pepsins A and C (also known as gastricsin).
Vertebrate chymosin (rennin), involved in digestion and used for making cheese.
Vertebrate lysosomal cathepsins D (EC 3.4.23.5) and E (EC 3.4.23.34).
Mammalian renin (EC 3.4.23.15) whose function is to generate angiotensin I from angiotensinogen in the plasma.
Fungal proteases such as aspergillopepsin A (EC 3.4.23.18), candidapepsin (EC 3.4.23.24), mucoropepsin (EC 3.4.23.23) (mucor rennin), endothiapepsin (EC 3.4.23.22), polyporopepsin (EC 3.4.23.29), and rhizopuspepsin (EC 3.4.23.21).
Yeast saccharopepsin (EC 3.4.23.25) (proteinase A) (gene PEP4). PEP4 is implicated in posttranslational regulation of vacuolar hydrolases.
Yeast barrierpepsin (EC 3.4.23.35) (gene BAR1), a protease that cleaves alpha-factor and thus acts as an antagonist of the mating pheromone.
Fission yeast sxa1 which is involved in degrading or processing the mating pheromones.
Most retroviruses and some plant viruses, such as badnaviruses, encode an aspartyl protease which is an homodimer of a chain of about 95 to 125 amino acids. In most retroviruses, the protease is encoded as a segment of a polyprotein which is cleaved during the maturation process of the virus. It is generally part of the pol polyprotein and, more rarely, of the gag polyprotein.
Caspase Family Active Sites
Interleukin-1 beta converting enzyme (EC 3.4.22.36) (ICE) (Thornberry et al. (1995)
Protein Sci
. 4:3-12; Kumar (1995)
Trends Biochem. Sci
. 20:198-202) is responsible for the cleavage of the IL-1 beta precursor at an Asp-Ala bond to generate the mature biologically active cytokine, ICE, a thiol protease composed of two subunits of 10 (p10) and 20 Kd (p20), both derived by the autocleavage of a 45 Kd precursor (p45). Two residues
Achutamurthy Ponnathapu
Alston & Bird LLP
Millennium Pharmaceuticals Inc.
Moore William W.
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