Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-11-28
2003-09-30
Kunz, Gary (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S388260, C530S389100, C530S389200
Reexamination Certificate
active
06627739
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the discovery of various active forms of &bgr;-secretase, an enzyme that cleaves &bgr;-amyloid precursor protein (APP) at one of the two cleavage sites necessary to produce &bgr;-amyloid peptide (A&bgr;). The invention also relates to inhibitors of this enzyme, which are considered candidates for therapeutics in the treatment of amyloidogenic diseases such as Alzheimer's disease. Further aspects of the present invention include screening methods, assays, and kits for discovering such therapeutic inhibitors, as well as diagnostic methods for determining whether an individual carries a mutant form of the enzyme.
BACKGROUND OF THE INVENTION
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillatory tangles present in the brain, particularly in those regions of the brain involved in memory and cognition. &bgr;-amyloid peptide (A&bgr;) is a 39-43 amino acid peptide that is major component of amyloid plaques and is produced by cleavage of a large protein known as the amyloid precursor protein (APP) at a specific site(s) within the N-terminal region of the protein. Normal processing of APP involves cleavage of the protein at point 16-17 amino acids C-terminal to the N-terminus of the &bgr;-AP region, releasing a secreted ectodomain, &agr;-sAPP, thus precluding production of &bgr;-AP. Cleavage by &bgr;-secretase enzyme of APP between Met
671
and Asp
672
and subsequent processing at the C-terminal end of APP produces A&bgr; peptide, which is highly implicated in the etiology of Alzheimer's pathology (Seubert, et al., in Pharmacological Treatment of Alzheimer's disease, Wiley-Liss, Inc., pp. 345-366, 1997; Zhao, J., et al. J. Biol. Chem. 271: 31407-31411, 1996).
It is not clear whether &bgr;-secretase enzyme levels and/or activity is inherently higher than normal in Alzheimer's patients; however, it is clear that its cleavage product, A&bgr; peptide, is abnormally concentrated in amyloid plaques present in their brains. Therefore, it would be desirable to isolate, purify and characterize the enzyme responsible for the pathogenic cleavage of APP in order to help answer this and other questions surrounding the etiology of the disease. In particular, it is also desirable to utilize the isolated enzyme, or active fragments thereof, in methods for screening candidate drugs for ability to inhibit the activity of &bgr;-secretase. Drugs exhibiting inhibitory effects on &bgr;-secretase activity are expected to be useful therapeutics in the treatment of Alzheimer's disease and other amyloidogenic disorders characterized by deposition of A&bgr; peptide containing fibrils.
U.S. Pat. No. 5,744,346 (Chrysler, et al.) describes the initial isolation and partial purification of &bgr;-secretase enzyme characterized by its size (apparent molecular weight in the range of 260 to 300 kilodaltons when measured by gel exclusion chromatography) and enzymatic activity (ability to cleave the 695-amino acid isotype of &bgr;-amyloid precursor protein between amino acids 596 and 597). The present invention provides a significant improvement in the purity of &bgr;-secretase enzyme, by providing a purified &bgr;-secretase enzyme that is at least 200 fold purer than that previously described. Such a purified protein has utility in a number of applications, including crystallization for structure determination. The invention also provides methods for producing recombinant forms of &bgr;-secretase enzymes that have the same size and enzymatic profiles as the naturally occurring forms. It is a further discovery of the present invention that human &bgr;-secretase is a so-called “aspartyl” (or “aspartic”) protease.
SUMMARY OF THE INVENTION
This invention is directed to a &bgr;-secretase protein that has now been purified to apparent homogeneity, and in particular to a purified protein characterized by a specific activity of at least about 0.2×10
5
and preferably at least 1.0×10
5
nM/h/&mgr;g protein in a representative &bgr;-secretase assay, the MBP-C125sw substrate assay. The resulting enzyme, which has a characteristic activity in cleaving the 695-amino acid isotype of &bgr;-amyloid precursor protein (&bgr;-APP) between amino acids 596 and 597 thereof, is at least 10,000-fold, preferably at least 20,000-fold and, more preferably in excess of 200,000-fold higher specific activity than an activity exhibited by a solubilized but unenriched membrane fraction from human 293 cells, such as have been earlier characterized.
In one embodiment, the purified enzyme is fewer than 450 amino acids in length, comprising a polypeptide having the amino acid sequence SEQ ID NO: 70 [63-452]. In preferred embodiments, the purified protein exists in a variety of “truncated forms” relative to the proenzyme reed to herein as SEQ ID NO: 2 [1-501], such as forms having amino acid sequences SEQ ID NO: 70 [63-452], SEQ ID NO: 69 [63-501], SEQ ID NO: 67 [58-501], SEQ ID NO: 68 [58-452], SEQ ID NO: 58 [46-452], SEQ ID NO: 74 [22-452], SEQ NO: 58 [46-452]. More generally, it has been found that particularly useful forms of the enzyme, particularly with regard to the crystallization studies described herein, are characterized by an N-terminus at position 46 with respect to SEQ ID NO: 2 and a C-terminus between positions 452 and 470 with respect to SEQ ID NO: 2. and more particularly, by an N-terminus at position 22 with respect to SEQ ID NO: 2 and a C-terminus between positions 452 and 470 with respect to SEQ ID NO: 2. These forms are considered to be cleaved in the transmembrane “anchor” domain. Other particularly useful purified forms of the enzyme include: SEQ ID NO: 43 [46-501], SEQ ID NO: 66 [22-501], and SEQ ID NO: 2 [1-501]. More generally, it is appreciated that useful forms of the enzyme have an N-terminal residue corresponding to a residue selected from the group consisting of residues 22, 46, 58 and 63 with respect to SEQ ID NO: 2 and a C-terminus selected from a residue between positions 452 and 501 with respect to SEQ ID NO: 2 or a C-terminus between residue positions 452 and 470 with respect to SEQ ID NO: 2. Also described herein are forms of enzyme isolated from a mouse, exemplified by SEQ ID NO: 65.
This invention is further directed to a crystalline protein composition formed from a purified &bgr;-secretase protein, such as the various protein compositions described above. According to one embodiment, the purified protein is characterized by an ability to bind to the &bgr;-secretase inhibitor substrate P10-P4′sta D→V which is at least equal to an ability exhibited by a protein having the amino acid sequence SEQ ID NO: 70 [46-4191], when the proteins are tested for binding to said substrate under the same conditions. According to another embodiment, the purified protein forming the crystallization composition is characterized by a binding affinity for the &bgr;-secretase inhibitor substrate SEQ ID NO: 72 (P10-P4′sta D→V) which is at least 1/100 of an affinity exhibited by a protein having the amino acid sequence SEQ ID NO: 43 [46-501], when said proteins are tested for binding to said substrate under the same conditions. Proteins forming the crystalline composition may be glycosylated or deglycosylated.
The invention also includes a crystalline protein composition containing a &bgr;-secretase substrate or inhibitor molecule, examples of which are provided herein, particularly exemplified by peptide-derived inhibitors such as SEQ ID NO: 78, SEQ ID NO: 72, SEQ ID NO: 81, and derivatives thereof. Generally useful inhibitors in this regard will have a K
i
of no more than about 50 &mgr;M to 0.5 mM.
Another aspect of the invention is directed to an isolated protein, comprising a polypeptide that (i) is fewer than about 450 amino acid residues in length, (ii) includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 75 [63
Anderson John P.
Basi Guriqbal
Doan Minh Tam
Frigon Normand
John Varghese
Elan Pharmaceuticals Inc.
Gucker Stephen
Kunz Gary
Townsend and Townsend / and Crew LLP
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