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WO2000068266A1 - Amyloid precursor protein protease and related nucleic acid compounds - Google Patents

Amyloid precursor protein protease and related nucleic acid compounds Download PDF

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Publication number
WO2000068266A1
WO2000068266A1 PCT/US2000/006707 US0006707W WO0068266A1 WO 2000068266 A1 WO2000068266 A1 WO 2000068266A1 US 0006707 W US0006707 W US 0006707W WO 0068266 A1 WO0068266 A1 WO 0068266A1
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seq
leu
val
ala
gly
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French (fr)
Inventor
Gerald Wayne Becker
John Edward Hale
William Francis Heath, Jr.
Edward Marion Johnstone
Sheila Parks Little
Yuan Tu
Wu-Kuang Yeh
Tinggui Yin
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Eli Lilly and Co
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to proteases which cleave amyloid precursor protein and facilitate ⁇ -amyloid peptide release, and accordingly, have utility in identifying compounds which treat or prevent Alzheimer's Disease.
  • Alzheimer's disease is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately death.
  • Alzheimer's disease is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States.
  • Alzheimer's disease has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. -Alzheimer's disease is at present incurable. No treatment that effectively prevents Alzheimer's disease or reverses its symptoms and course is currently known.
  • the brains of individuals with Alzheimer's disease exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles.
  • senile or amyloid
  • amyloid angiopathy amyloid deposits in blood vessels
  • neurofibrillary tangles Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with Alzheimer's disease. Smaller numbers of these lesions in a more restrictive anatomical distribution are also found in the brains of most aged humans who do not have clinical Alzheimer's disease.
  • Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D).
  • amyloid angiopathy The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy), characteristic of Alzheimer's disease and the other disorders mentioned above, is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the ⁇ -amyloid peptide (this protein is also referred to in the literature as ⁇ AP, A ⁇ , A ⁇ P, A- beta, or ⁇ /A4).
  • ⁇ -Amyloid peptide was first purified and a partial amino acid sequence was provided by Glenner, et al.. Biochemical and Biophysical Research Communications, 120:885-890 (1984). The isolation procedure and the sequence data for the first 28 amino acids are described in United States Patent 4,666,829, the entire contents of which are herein incorporated by reference.
  • ⁇ -amyloid peptide is a small fragment of a much larger precursor protein, the amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans.
  • APP amyloid precursor protein
  • Knowledge of the structure of the gene encoding the amyloid precursor protein has demonstrated that the ⁇ -amyloid peptide arises as a peptide fragment that is cleaved from the amyloid precursor protein by proteases.
  • a normal (i.e., non-pathogenic) processing of the amyloid precursor protein occurs via cleavage by a putative " -secretase” which cleaves between amino acids 16 and 17 of the ⁇ -amyloid peptide region within the protein. Kang, et al., Nature (London), 325:773-776 (1987). It is further believed that pathogenic processing occurs in part via an enzyme designated " ⁇ -secretase" which cleaves at the amino-terminus of the ⁇ -amyloid peptide region within the precursor protein.
  • the identification, isolation, and purification of the proteases involved in the processing of amyloid precursor protein would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit formation of ⁇ -amyloid peptide.
  • the treatment methods would advantageously be based on drugs which are capable of inhibiting ⁇ -amyloid peptide release and/or its synthesis in vivo.
  • the present invention provides an isolated protein useful as an amyloid precursor protein protease, said compound comprising the amino acid sequence
  • SEQ ID NO:2 525 530 535 hereinafter referred to as SEQ ID NO:2.
  • amyloid precursor protein protease comprising the amino acid sequence Met Leu Arg Arg Arg Gly Ser Pro Gly 1 5
  • SEQ ID NO:4 525 530 535 hereinafter referred to as SEQ ID NO:4.
  • the invention also provides isolated nucleic acid compounds that comprise nucleic acid sequences which encode the amino acid compounds provided.
  • the isolated nucleic acid compounds that are provided are preferably DNA, however, nucleic acid compounds which are sense or antisense mRNA are also provided.
  • a particularly preferred nucleic acid compound is the DNA compound comprising the sequence
  • cag ctg gtg cac age ttt get gag ggc cag gac cag ggc age gcc tat 291 Gin Leu Val His Ser Phe Ala Glu Gly Gin Asp Gin Gly Ser Ala Tyr 75 80 85
  • gtg gcc get ccc tac teg aag ccc age atg ace ctg gag ccc aac aag 483 Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys 140 145 150
  • ctg act ggc aac gtg ace aeg teg cag atg gcc aac gag cag ggc ttg 627
  • aac etc ate tgg cag ctg aca gac ace aaa cag ctg gtg cac agt ttc 915 Asn Leu He Trp Gin Leu Thr Asp Thr Lys Gin Leu Val His Ser Phe 285 290 295
  • an even more preferred isolated nucleic acid compound is the DNA compound having the sequence agctgtcagc cgcctcacag gaag atg ctg cgt egg egg ggc age cct ggc 51
  • cag ctg gtg cac age ttt get gag ggc cag gac cag ggc age gcc tat 291
  • ggc tac cct gag get gag gtg ttc tgg cag gat ggg cag ggt gtg ccc 579 Gly Tyr Pro Glu Ala Glu Val Phe Trp Gin Asp Gly Gin Gly Val Pro 170 175 180 185
  • ctg act ggc aac gtg ace aeg teg cag atg gcc aac gag cag ggc ttg 627 Leu Thr Gly Asn Val Thr Thr Ser Gin Met Ala Asn Glu Gin Gly Leu
  • SEQ ID NO:3 SEQ ID NO:3.
  • nucleic acid vectors comprising nucleic acids which encode SEQ ID NO:2 or SEQ ID NO:4 or functional equivalents thereof.
  • the preferred nucleic acid vectors are those which are DNA.
  • DNA vectors of the present invention preferably comprise a promoter positioned to drive expression of said DNA sequence. Those vectors wherein said promoter functions in human embryonic kidney cells (293 cells), AV12 cells, yeast cells or E.coli cells are preferred.
  • the present invention also provides probes and primers useful for molecular biology techniques.
  • a compound which encodes all or part of SEQ ID NO:2 or SEQ ID NO:4 and which is at least 18 consecutive base pairs in length is provided.
  • the 18 base pair or more compound is DNA.
  • Most preferred for this use are the DNA compounds which comprise at least 18 consecutive base pairs of SEQ ID NO:l or SEQ ID NO:3.
  • a preferred host cell is an oocyte.
  • a preferred oocyte is one which has been injected with sense mRNA or DNA compounds of the present invention.
  • a more preferred oocyte is one which has been injected with sense mRNA or DNA compounds of the present invention in conjunction with DNA or sense mRNA which encodes APP.
  • this invention provides cells into which the nucleic acid compounds of the present invention may be transfected.
  • Host cells include those which are transfected with a nucleic acid compound which encodes SEQ ID NO:2 or SEQ ID NO:4.
  • Preferred cells include host cells transfected with a DNA vector comprising SEQ ID NO:l or SEQ ID NO:3.
  • the preferred transfected host cells which encode SEQ ID NO:2 or SEQ ID NO:4 are 293 cells, AV12 cells, yeast cells and E. coli cells.
  • the invention provides a method for identifying DNA homologous to a probe of the present invention, which comprises combining test nucleic acid with the probe under hybridizing conditions and identifying those test nucleic acids which hybridize.
  • Assays utilizing the compounds provided by the present invention are also provided.
  • the assays provided determine whether a substance is a ligand for ⁇ -secretase, said method comprising contacting ⁇ -secretase with said substance, monitoring ⁇ -secretase activity by physically detectable means, and identifying those substances which interact with or affect ⁇ -secretase.
  • Preferred assays of the present invention include a cell culture assay, a high-performance liquid chromotography (HPLC) assay or a synthetic competition assay.
  • Pre- ferred cell culture assays utilize oocytes, AN12, E. coli, yeast or 293 cells which co-express nucleic acids which encode ⁇ -secretase and APP.
  • the invention also provides methods for constructing a host cell capable of expressing a nucleic acid compound which encodes an amino acid compound comprising SEQ ID ⁇ O:2 or SEQ ID NO:4, said methods comprising transfecting a host cell with a DNA vector comprising a nucleic acid compound encoding SEQ ID NO:2 or SEQ ID NO:4.
  • a preferred method utilizes 293, AV12, yeast or E. coli cells as the host cells.
  • a more preferred method includes a DNA vector which comprises SEQ ID NO:l or SEQ ID NO:3.
  • a most preferred method includes a DNA vector which comprises SEQ ID NO:3.
  • Another preferred method comprises (a) a DNA vector which comprises SEQ ID NO: 3 and (b) a DNA expression vector which encodes the APP coding sequence.
  • methods for expressing a nucleic acid sequence which encodes SEQ ID NO:2 or SEQ ID NO:4, or functional equivalents thereof , in a transfected host cell comprise culturing a transfected host cell of the present invention under conditions suitable for gene expression.
  • a preferred method utilizes 293, AV12, yeast or E. coli cells as the transfected host cell.
  • a more preferred method utilizes a DNA vector to transfect the host cell.
  • a most preferred method utilizes a DNA vector comprising all or part of SEQ ID NO:3.
  • the present invention also provides processes for isolating and purifying an amyloid precursor protein protease, said process comprising: (a) establishing in a suitable medium, a culture of the host cells transformed with a polynucleotide encoding an amyloid precursor protein protease; and (b) isolating said protease from said culture.
  • compositions comprising a peptide isolated according to such processes are also provided.
  • polyclonal and monoclonal antibodies to the peptides of the present invention are also provided.
  • Base pair refers to DNA or RNA.
  • the abbreviations A, C, G, and T correspond to the 5'-monophosphate forms of the deoxyribonucleosides (deoxy)adenosine, (deoxy)cytidine, (deoxy)guanosine, and (deoxy)thymidine, respectively, when they occur in DNA molecules.
  • the abbreviations U, C, G, and A correspond to the 5'-monophosphate forms of the ribonucleosides uridine, cytidine, guanosine, and adenosine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • heteroduplex base pair may refer to a partnership of A with U or C with G. (See the definition of "complementary”, infra.)
  • restriction refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA ("sequence-specific endonucleases”).
  • restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can be readily found in the literature.
  • Ligation refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with a DNA ligase, such as T4 DNA ligase.
  • plasmid refers to an extrachromosomal (usually) self- replicating genetic element. Plasmids are generally designated by a lower case “p” preceded and/or followed by letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • reading frame means the nucleotide sequence from which translation occurs “read” in triplets by the translational apparatus of transfer RNA (tRNA) and ribosomes and associated factors, each triplet corresponding to a particular amino acid. To insure against improper translation, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame” being maintained.
  • Recombinant DNA cloning vector refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • recombinant DNA expression vector refers to any recombinant DNA cloning vector in which a promoter to control transcription of the inserted DNA has been incorporated.
  • expression vector system refers to a recombinant DNA expression vector in combination with one or more trans- acting factors that specifically influence transcription, stability, or replication of the recombinant DNA expression vector.
  • the trans-acting factor may be expressed from a co-transfected plasmid, virus, or other extrachromosomal element, or may be expressed from a gene integrated within the chromosome.
  • Transcription refers to the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
  • transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, calcium phosphate co-precipitation, and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
  • transformation means the introduction of DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.
  • Methods of transforming bacterial and eukaryotic hosts are well known in the art, many of which methods, such as nuclear injection, protoplast fusion or by calcium treatment using calcium chloride are summarized in J. Sambrook, et al.. MOLECULAR CLONING: A LABORATORY MANUAL, (1989).
  • transformation refers to the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.
  • vector refers to a nucleic acid compound used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which when combined with appropriate control sequences confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases.
  • vector as used herein includes Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
  • complementary refers to the capacity of p urine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules.
  • the following base pairs are related by complementarity: guanine and cytosine; adenine and thymine; and adenine and uracil.
  • complementary means that the aforementioned relationship applies to substantially all base pairs comprising two single-stranded nucleic acid molecules over the entire length of said molecules.
  • Partially complementary refers to the aforementioned relationship in which one of two single-stranded nucleic acid molecules is shorter in length than the other such that a portion of one of the molecules remains single-stranded.
  • hybridization refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing.
  • the conditions employed in the hybridization of two completely or nearly completely complementary nucleic acid strands varies with the degree of complementarity of the two strands and the length of the strands. Such techniques and conditions are well known to practitioners in this field.
  • isolated amino acid sequence refers to any amino acid sequence, however constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
  • isolated DNA compound refers to any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
  • isolated nucleic acid compound refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.
  • a "primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation.
  • promoter refers to a DNA sequence which directs transcription of DNA to RNA.
  • a “probe” as used herein is a nucleic acid compound or a fragment thereof which hybridizes with a nucleic acid compound which encodes either the entire sequence of SEQ ID NO:2 or SEQ ID NO:4, a sequence complementary to SEQ ID NO:2 or SEQ ID NO:4, or a part thereof.
  • EST or “expressed sequence tag” refers to a fragment or sampling of cDNA which encodes a polypeptide of unknown function.
  • stringency refers to a set of hybridization conditions which may be varied in order to vary the degree of nucleic acid affinity for other nucleic acid. (See the definition of "hybridization”, supra.)
  • telomere sequence refers to a sequence in which antibodies raised against an epitope of the proteins of the present invention, or a fragment thereof, may be used to differentiate between the proteins of the present invention and other ⁇ -secretase variants. This term may also be employed in the sense that such antibodies may be used to differentiate between the human ⁇ -secretase protein and analogous proteins derived from other species.
  • PCR refers to the widely-known polymerase chain reaction employing a thermally-stable polymerase. This technique, as appreciated by those skilled in the art, is employed to amplify a particular nucleic acid fragment.
  • BLAST refers to the widely known basic local alignment search tool.
  • This tool consists of a set of computer- based programs designed to permit examination of amino acid and nucleic acid sequence databases for similarity with an isolated sequence of interest.
  • RACE refers to the widely known rapid amplification of complimentary ends technique, to amplify and obtain the 5' and 3' ends of isolated cDNA.
  • proteins of the present invention can be synthesized by a number of different methods. All of the amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis, or recombinant methods. Both methods are described in U.S. Patent 4,617,149, herein incorporated by reference.
  • peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems.
  • Protected amino acids such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses. Sequential t-butoxycarbonyl chemistry using double couple protocols are applied to the starting /-.-methyl benzhydryl amine resins for the production of C-terminal carboxamides.
  • pyridine-2-aldoxime methiodide resin For the production of C-terminal acids , the corresponding pyridine-2-aldoxime methiodide resin is used. Asparagine, glutamine, and arginine are coupled using preformed hydroxy benzotriazole esters. The following side chain protection may be used:
  • the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees centigrade or below, preferably - 20°C for thirty minutes followed by thirty minutes at 0°C.
  • the proteins of the present invention may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired.
  • a general method for the construction of any desired DNA sequence is provided in J. Brown, et al., Methods in Enzymology, 68:109 (1979). See also, J. Sambrook, et al., supra. The basic steps in the recombinant production of desired proteins are:
  • prokaryotes are used for cloning of DNA sequences in constructing the vectors of this invention.
  • Prokaryotes may also be employed in the production of the protein of interest.
  • the Escherichia coli K12 strain 294 ATCC No. 31446
  • Other strains of E. coli which may be used (and their relevant genotypes) include the following.
  • DH5a F- (j80dlacZDMl5), D(lacZYA-argF)U169 supE44, l-> hsdR17(r K -, m ⁇ + ), recAl, endAl, gyrA96, thi-1, relAl
  • strains are all commercially available from suppliers such as: Bethesda Research Laboratories, Gaithersburg, Maryland 20877 and Stratagene Cloning Systems, La Jolla, California 92037; or are readily available to the public from sources such as the American Type Culture
  • genotypes listed are illustrative of many of the desired characteristics for choosing a bacterial host and are not meant to limit the invention in any way.
  • the genotype designations are in accordance with standard nomenclature. See, for example, J. Sambrook, et al., supra.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella tvphimurium or Serratia marcescans
  • various Pseudomonas species may be used.
  • other bacteria especially Streptomvces, spp., may be employed in the prokaryotic cloning and expression of the proteins of this invention.
  • Promoters suitable for use with prokaryotic hosts include the b- lactamase [vector pGX2907 (ATCC 39344) contains the replicon and b- lactamase gene] and lactose promoter systems [Chang et al.. Nature (London), 275:615 (1978); and Goeddel et al.. Nature (London), 281:544
  • alkaline phosphatase alkaline phosphatase
  • the tryptophan (trp) promoter system vector pATHl (ATCC 37695) is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter] and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC- 37282).
  • trp tryptophan
  • vector pATHl ATCC 37695
  • hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC- 37282).
  • other functional bacterial promoters whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine -Dalgarno sequence operably linked to the DNA encoding the desired polypeptide
  • the proteins of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the lifespan, increases the yield of the desired peptide, or provides a convenient means of purifying the protein of interest.
  • a variety of peptidases e.g. trypsin which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known.
  • particular chemicals e.g.
  • cyanogen bromide will cleave a polypeptide chain at specific sites.
  • the skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13 in PROTEIN PURIFICATION: FROM MOLECULAR MECHANISMS TO LARGE SCALE PROCESSES, American Chemical Society, Washington, DC (1990).
  • the proteins of the present invention may also be produced in eukaryotic systems.
  • the present invention is not limited to use in a particular eukaryotic host cell.
  • a variety of eukaryotic host cells are available from depositories such as the American Type Culture Collection (ATCC) and are suitable for use with the vectors of the present invention.
  • ATCC American Type Culture Collection
  • the choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of the amyloid precursor protein protease-encoding nucleic acids of the present invention.
  • Exemplary host cells suitable for use in the present invention are listed in Table I. Theses exemplary host cells are merely illustrative of the many eukaryotic cells available for use with the present invention and are not meant in any way to limit the scope of the present invention.
  • a preferred cell line employed in the expression of the protein of the present invention is the widely available 293 cell line. As noted, this cell line was constructed from human embryonal kidney tissue and is available from American Type Culture Collection under the accession number ATCC CCL 1573.
  • Some illustrative vectors include the pSV2-type vectors which comprise segments of the simian virus 40 (SN40) genome that constitute a defined eukaryotic transcription unit-promoter, intervening sequence, and polyadenylation site.
  • SN40 simian virus 40
  • the plasmid pSN2-type vectors transform mammalian and other eukaryotic host cells by integrating into the host cell chromosomal D ⁇ A.
  • Plasmid pSV2-type vectors such as plasmid pSN2-gpt, pSN2- neo, pSN2-dhfr, pSN2-hyg, and pSV2-b-globin, in which the SN40 promoter drives transcription of an inserted gene.
  • These vectors are suitable for use with the coding sequences of the present invention and are widely available from sources such as the ATCC or the Northern Regional Research Laboratory (NRRL), 1815 N. University Street, Peoria, Illinois, 61604.
  • the plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolate reductase (dhfr) gene under the control of the SV40 early promoter. Under the appropriate conditions, the dhfr gene is known to be amplified, or copied, in the host chromosome. This amplification can result in the amplification of closely-associated DNA sequences and can, therefore, be used to increase production of a protein of interest. See, e.g., J. Schimke, Cell, 35:705-713 (1984). Plasmids constructed for expression of the proteins of the present invention in mammalian and other eukaryotic host cells can utilize a wide variety of promoters.
  • the present invention is in no way limited to the use of the particular promoters exemplified herein.
  • Promoters such as the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene, and the major early and late adenovirus genes can be readily isolated and modified to express the genes of the present invention.
  • Eukaryotic promoters can also be used in tandem to drive expression of a coding sequence of this invention.
  • Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention. H. Gorman, et al., Proceedings of the National Academy of Sciences (USA). 79:6777 (1982).
  • the plasmid pMSNi (NRRL B- 15929) comprises the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells.
  • the mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the expression of the nucleic acids of the present invention.
  • the mouse metallothionein promoter is present in the plasmid pdBPN-MMTneo (ATCC 37224) which can serve as the starting material of other plasmids of the present invention.
  • Another usefull expression vector system employs one of a series of vectors containing the BK enhancer, an enhancer derived from the BK virus, a human papovavirus.
  • the most preferred such vector systems are those which employ not only the BK enhancer but also the adenovirus-2-early region IA (El A) gene product.
  • the E1A gene product (actually, the E1A gene produces two products, which are collectively referred to herein as "the E1A gene product”) is an immediate-early gene product of adenovirus, a large D ⁇ A virus.
  • An additional useful eukaryotic expression vector is the phd series of vectors which comprise a BK enhancer in tandem with the adenovirus late promoter to drive expression of useful products in eukaryotic host cells.
  • the construction and method of using the phd plasmid, as well as related plasmids, are described in U.S. Patents 5,242,688, issued September 7, 1993, and 4,992,373, issued February 12, 1991, all of which are herein incorporated by reference.
  • Escherichia coli K12 GM48 cells harboring the plasmid phd are available as part of the permanent stock collection of the Northern Regional Research Laboratory under accession number NRRL B- 18525. The plasmid may be isolated from this culture using standard techniques.
  • the plasmid phd contains a unique Bell site which may be utilized for the insertion of the gene encoding the protein of interest.
  • the skilled artisan understands that linkers or adapters may be employed in cloning the gene of interest into this Bell site.
  • the phd series of plasmids functions most efficiently when introduced into a host cell which produces the E1A gene product, cell lines such as AN12-664, 293 cells, and others, described supra.
  • Transformation of the mammalian cells can be performed by any of the known processes including, but not limited to, the protoplast fusion method, the calcium phosphate co-precipitation method, electroporation and the like. See, e.g., J. Sambrook, et al., supra, at 3:16.30-3:16.66.
  • viruses are also appropriate vectors.
  • the adenovirus, the adeno- associated virus, the vaccinia virus, the herpes virus, the baculovirus, and the rous sarcoma virus are useful.
  • Such a method is described in U.S. Patent 4,775,624, herein incorporated by reference.
  • Several alternate methods of expression are described in J. Sambrook, et al., supra, at 16.3-17.44.
  • eukaryotic microbes such as yeast cultures may also be used.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • the plasmid YRp7 ATCC-40053
  • yeast cultures may also be used. See, e.g., L. Stinchcomb, et al., Nature (London), 282:39 (1979); J. Kingsman et al.. Gene, 7:141 (1979); S.
  • This plasmid already contains the trp_ gene which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [found on plasmid pAP12BD (ATCC 53231) and described in U.S. Patent No. 4,935,350, issued June 19, 1990, herein incorporated by reference] or other glycolytic enzymes such as enolase [found on plasmid p AC 1 (ATCC 39532)], glyceraldehyde-3-phosphate dehydrogenase [derived from plasmid pHcGAPCl (ATCC 57090, 57091)], hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, p ho sp ho glucose isomerase, and glucokinase, as well as the alcohol dehydrogenase and pyru
  • yeast promoters which are inducible promoters, having the additional advantage of their transcription being controllable by varying growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein [contained on plasmid vector
  • P CL28XhoLHBPV (ATCC 39475) and described in United States Patent No. 4,840,896, herein incorporated by reference], glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose [e.g. GAL1 found on plasmid pRY121 (ATCC 37658)] utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in R.
  • Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjunction with the CYC1 promoter on plasmid YEpsec-hllbeta ATCC 67024), also are advantageously used with yeast promoters.
  • Practitioners of this invention realize that, in addition to the above-mentioned expression systems, cloned cDNA may also be employed in the production of transgenic animals in which a test mammal, usually a mouse, in which expression or overexpression of the proteins of the present invention can be assessed.
  • the nucleic acids of the present invention may also be employed in the construction of "knockout" animals in which the expression of the native cognate of the gene is suppressed.
  • protease of the present invention can be isolated and purified from cultured host cells transiently expressing ⁇ - secretase activity.
  • the following examples further illustrate such isolation and purification processes, however, these examples are not in any way to be interpreted as limiting the scope of the present invention.
  • the enzymatic activity of beta-secretase was determined by an ELISA assay, modified in form over an assay appearing in application No. PCT/US96/09985, the entire content of which is herein incorporated by reference.
  • MBP-APPcl25sw as the substrate, the activity was determined by measuring the MBP-APPc26sw cleavage product with a polyclonal antibody, 192sw, highly specific to the product.
  • MBP-APPcl25sw is a fusion substrate in which maltose binding protein is fused to the 125- residue C-terminal portion of the Swedish amyloid precursor protein.
  • one picounit is defined as the amount of the enzyme that catalyzes the conversion of MBP-APP125sw to one picomole of MBP-APPc26sw per ml in 2-hr under the optimized reaction conditions of the Elisa assay (see Table II, infra).
  • the protein concentration was first determined using the well known Bio-Rad microassay method. (Rio-Rad Laboratories, Hercules, CA, 94547) Example 3 ⁇ -Secretase Purification
  • beta-secretase was carried out by a protocol modified over the purification methods appearing in application No. PCT/US96/09985, the entire content of which is herein incorporated by reference.
  • the enzyme purification protocol was undertaken at 4 C.
  • ⁇ - Secretase activity and protein recovery data are provided in Table III, infra.
  • Membrane Extract About 400-g of 293 cells were resuspended in 2,000-ml of 20 mM HEPES (Sigma Chemical Co., St Louis, MO), pH 7.5, 2 mM EDTA (Sigma Chemical Co., St Louis, MO) and 0.25% sucrose (Buffer IA). The cell suspension was homogenized in an 1000-ml aliquot and the cells were broken with an Brinkmann Homogenizer (POLYTRON, Switzerland) according to the following conditions: at setting 4 for 2- and 1-min; and at setting 5 for 1- and 0.5-min (with 3-min cooling time after each homogenization).
  • Brinkmann Homogenizer POLYTRON, Switzerland
  • the broken cell suspension was centrifuged at l,000xg for 20-min; the resulting supernatant (post-nuclear supernatant; PNSl) was saved.
  • the pellet was resuspended in 1,200-ml of Buffer IA, homogenized , broken and centrifuged as described above; the supernatant (PNS2) was saved and the pellet was discarded.
  • PNSl and PNS2 were combined (2,700-ml) and centrifuged at 45,000xg for 1-hr; the resulting pellet (P2; containing membranes) was saved and the supernatant was discarded.
  • This pellet was resuspended in 3,200-ml of 20 mM MES (Sigma Chemical Co., St Louis, MO), pH 6.0, 2 mM EDTA, 0.5% Triton X-100, 150 mM NaCl, 0.2 mM AEBSF, 5 mg/ml leupeptin, 2 mg/ml E64 and 1 mg/ml pepstatin A (Buffer IB); all four protease inhibitors were from Calbiochem; San Diego, CA).
  • the suspension was stirred continuously for 1-hr for extraction of beta-secretase and centrifuged at 16,000xg for 1-hr; the pellet was discarded.
  • the resulting supernatant (3,120- ml) was adjusted to pH 7.5 with 1 M Tris base and filtered through 0.45-mm Zapcap-S (Schleicher & Schuell; Keene, NH); the filtrate was the P2 membrane extract (P2ME, 3,170-ml).
  • a second P2ME was prepared similarly from another 400g of 293 cells.
  • the combined beta-secretase activity and protein data for the P2ME fractions are shown in Table III.
  • beta-secretase activity from each P2ME was purified separately in the next step.
  • WGA Wheat Germ Agglutinin
  • the first P2ME was loaded to a WGA-Agarose column (Vector Lab.; Burlingame, CA) (1000-ml), pre-equilibrated with 1500-ml of 20 mM Tris, pH 7.5, 2 mM EDTA, 0.5% Triton X-100 and 150 mM NaCl (Buffer 2A).
  • the WGA column which binds to N-acetyl-D-glucosamine glycopeptides, was washed with 1000-ml of Buffer 2A and bound proteins were eluted with a linear gradient of 0-10% chitin hydrolysate (Vector Lab.) in a total volume of 900-ml constructed from Buffer 2A and Buffer 2B (20 mM Tris, pH 7.5, 2 mM EDTA, 0.5% Triton X-100 and 10% chitin hydrolysate). The fractions containing higher specific activities of beta-secretase were pooled as the WGA-eluate (180-ml).
  • the WGA-Agarose column was regenerated and equilibrated.
  • the second WGA-eluate (170-ml) was obtained similarly from the second P2ME.
  • the two WGA-eluates were combined. (see Table III).
  • the two WGA- Agarose washings were combined. (see Table III).
  • the WGA-eluate and the WGA-wash were purified together in the next step.
  • HiTrap Q (HiQ) Eluate The WGA-eluate and the WGA-wash were diluted 6x and 4x, respectively, with 20 mM Tris, pH 8.0, 2 mM EDTA and 0.2% Triton X-100 (Buffer 3A) to reduce Triton X-100 and NaCl concentrations. Both diluted enzyme preparations were loaded consecutively to four connected, fast flow anion exchanger HiTrap Q columns (4x5-ml; Amersham Pharmacia Biotech, Piscataway, NJ), pre-equilibrated with 100-ml of Buffer 3A, and the column was washed with 200-ml of Buffer 3A.
  • Bound proteins were eluted first with a linear gradient of 0-500 mM NaCl in buffer 3A in a total volume of 420-ml and then eluted with 100-ml of Buffer 3B (Buffer 3A plus 500 mM NaCl). The fractions containing higher specific activities were pooled as the HiQ-eluate. (Table III) The HiQ-eluate was used directly in the next step.
  • HA Hydroxylapatite Wash: The HiQ-eluate was loaded to a Hydroxylapatite column (10-ml; Bio-Rad, Hercules, CA), pre-equilibrated with 100-ml of Buffer 4A (same as Buffer 3A), the column flow-through was collected. HA separation of proteins is based on charge absorption and desorption. The column was washed with 50-ml of Buffer 4A, and the bound proteins were eluted from the column with a linear gradient of 0-500 mM potassium phosphate in Buffer 4A in a total volume of 100-ml. Among the three fractions (the flow-through, the wash, and the eluate), the HA-wash (50-ml) showed the highest total , and specific activity of beta-secretase (Table III) and, thus, was used in the next step.
  • the HA-wash was de-salted to about 15 mM NaCl with Buffer 5A (same as Buffer 4A) using Centriprep 50 (Millipore, Bedford, MA ).
  • the de-salted HA-wash was loaded to a high resolving strong anion exchanger Mono Q column (1-ml; Amersham Pharmacia Biotech), pre- equilibrated with 30-ml of Buffer 5A.
  • the column was washed with 30-ml of buffer 5A, and bound proteins were eluted first with a linear gradient of 0- 500 mM in Buffer 5A in a total volume of 20-ml and then eluted with 10-ml of Buffer 5B (Buffer 5A plus 500 mM NaCl). Two activity peaks were observed and the active fractions from each activity peak were pooled. (Table III) The two Mono Q-eluates (2-ml & 4-ml)were purified separately in the next step.
  • Mono Q-eluate 1 was loaded to a high resolution gel-filtration Superdex 200 column (300-ml; Amersham Pharmacia Biotech), pre-equilibrated with 900-ml of 20 mM Tris, pH 8.0, 2 mM EDTA, 0.2% Triton X-100, 1 mM MgCl 2 , 1 mM CaCl 2 and 150 mM NaCl (Buffer 6).
  • the proteins were eluted with 300-ml of Buffer 6. Two activity peaks were observed and the active fractions from both peaks were pooled together (S- 200-eluate 1, 19.2-ml)(Table III) and used in the next step.
  • Mono Q-eluate 2 was similarly purified by the same Superdex 200 column; three activity peaks were observed.
  • the two Superdex 200-eluates that showed the higher specific activities (S-200 eluates 2a & 2b; Table III) were used separately in the next step.
  • the bound proteins were eluted from DEAE-Sepharose beads of the three samples with, respectively, 2x200-, 2x400- and 2x100 ⁇ l of Buffer 7B (Buffer 7A plus 1 M NaCl).
  • Buffer 7B Buffer 7A plus 1 M NaCl.
  • the three DEAE-eluates 400-, 800-, and 200 ⁇ l; Table III) were treated separately in the next step.
  • Buffer 9 A (same as Buffer 7A).
  • the bound proteins were washed and eluted with a step-wise NaCl gradient in Buffer 7A in a total volume of 35.6-ml as shown below: 7.6-ml at 0 mM NaCl, 16-ml from 0 to 250 mM, 8-ml from 250 to 500 mM and 4-ml at 500 mM. Two activity peaks were observed. Desalted De-Gly 2a were purified in a similar way; two activity peaks were also observed.
  • Desalted De-Gly 2b sample was purified with a Mini Q PC column (0.24-ml from Amersham Pharmacia Biotech) using a step-wise NaCl gradient in Buffer 7A in a total volume of 13.2-ml (4.8-ml at 0 mM NaCl, 4.8- ml from 0 to 250 mM, 2.4-ml from 250 to 500 mM and 1.2-ml at 500 mM). Only one major activity peak was observed. The resulting beta-secretase activity and protein data are shown in Table III.
  • Beta-secretase activity was purified up to 3, 000-fold to apparent homogeneity by the 9-step procedure. Note, however, that the actual activity enrichment was likely much greater excluding Triton X-100 interference to the low level protein determination in Step 9 above.
  • Mini Q-Eluate 2a represents the first peak from fraction Q-E la combined with the first peak from fraction
  • Mini Q-Eluate 2b represents the second peak from fraction Q-E la combined with the second peak from fraction Q-E lb.
  • SEQ ID NO: 2 or SEQ ID NO: 4 will fail to change the function of the amino acid compound.
  • some hydrophobic amino acids may be exchanged for other hydrophobic amino acids.
  • Those altered amino acid compounds which confer substantially the same function in substantially the same manner as the exemplified amino acid compounds are also encompassed within the present invention.
  • Typical such conservative substitutions attempt to preserve the: (a) secondary or tertiary structure of the polypeptide backbone; (b) the charge or hydrophobicity of the residue; or (c) the bulk of the side chain.
  • Some examples of such conservative substitutions of amino acids, resulting in the production of proteins which are functional equivalents of the protein of SEQ ID NO:2 or SEQ ID NO:4 are shown in Table II, infra.
  • substitutions may be introduced into the protein in a variety of ways, such as during the chemical synthesis or by chemical modification of an amino acid side chain after the protein has been prepared.
  • Alterations of the protein having a sequence which corresponds to the sequence of SEQ ID NO:2 or SEQ ID NO:4 may also be induced by alterations of the nucleic acid compounds which encodes these proteins. These mutations of the nucleic acid compound may be generated by either random mutagenesis techniques, such as those techniques employing chemical mutagens, or by site-specific mutagenesis employing oligonucleotides. In addition, allelic variants of the gene encoding the protein of the present invention may also be purified by the processes of the present invention. Those nucleic acid compounds which confer substantially the same function in substantially the same manner as the exemplified nucleic acid compounds are also encompassed within the present invention.
  • nucleic acid compounds which comprise isolated nucleic acid sequences which encode SEQ ID NO:2 or SEQ ID NO:4.
  • amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one nucleic acid triplet due to the degeneracy of the amino acid code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.
  • N- terminal fragments of the determined protein sequence were then searched against protein sequence databases using commercially available BLAST methodology.
  • a BLAST analysis of the proprietary INCYTE database revealed a series of ESTs showing homology to the determined experimental sequence. The ESTs were subsequently assembled into a contiguous cDNA sequence.
  • RACE-ready cDNA (CLONTECH Laboratories, Palo Alto, CA, 94303) was used as a template to elongate and amplify the 5' end until a starting methionine was detected. PCR was then employed to generate the full length cDNA clone. E. coli were subsequently transformed with the full length cDNAs comprising SEQ ID NO:l and SEQ ID NO:3 in order to generate sufficient copies of the nucleic acids for sequence determination.
  • the gene encoding the amyloid precursor protein protease of the present invention may also be produced using synthetic methodology, which synthesis is well known in the art. See, e.g., E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana, Methods in Enzvmology. 68:109-151 (1979).
  • nucleic acid sequences corresponding to the gene encoding the amyloid precursor protein protease of the present invention can also be generated using conventional DNA synthesizing apparatuses such as the Applied Biosystems Model 380A or 380B DNA synthesizers, (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) These synthesizers employ phosphoramidite chemistry. In the alternative, the more traditional phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. See, e.g., M.J. Gait, ed., OLIGONUCLEOTIDE SYNTHESIS, A PRACTICAL APPROACH, (1984).
  • the synthetic amyloid precursor protein protease gene may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into expression and amplification plasmids.
  • the restriction sites are chosen so as to properly orient the coding sequence of the target enzyme with control sequences to achieve proper in-frame reading and expression of the amyloid precursor protein protease molecule.
  • a variety of other such cleavage sites may be incorporated depending on the particular plasmid constructs employed and may be generated by techniques well known in the art.
  • the desired DNA sequences can be generated using the polymerase chain reaction as described in U.S. Patent No. 4,889,818, which is herein incorporated by reference.
  • the present invention also encompasses ribonucleic acid compounds which have a sequence which encodes SEQ ID NO:2 or SEQ ID NO:4.
  • the present invention provides the RNA sequence represented by
  • SEQ ID NO:5 which is herein referred to as SEQ ID NO:5.
  • ribonucleic acids of the present invention may be prepared using the polynucleotide synthetic methods discussed supra or they may be prepared enzymatically using any one of various RNA polymerases to transcribe a DNA template.
  • This invention also provides nucleic acids, RNA or DNA, which are complementary to SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5
  • the present invention also provides probes and primers useful for molecular biology techniques.
  • the 18 or more base pair compound is DNA.
  • Primers and probes may be obtained by means well known in the art. For example, once a protein of interest is isolated, restriction enzymes and subsequent gel separation may be used to isolate the fragment of choice.
  • the term "selectively hybridize" as used herein may refer to either of two situations. In the first such embodiment of this invention, the nucleic acid compounds described supra hybridize to DNA or RNA encoding a human ⁇ -secretase protease under more stringent hybridization conditions than these same nucleic acid compounds would hybridize to nucleic acids encoding analogous ⁇ -secretases of another species. In the second such embodiment of this invention, these probes hybridize to the DNA or RNA encoding the ⁇ -secretase under more stringent hybridization conditions than other related compounds, including nucleic acid sequences encoding other amyloid precursor protein proteases.
  • probes and primers can be prepared enzymatically as described supra. In a most preferred embodiment, however, these probes and primers are synthesized using chemical means as described herein.
  • probes and primers are well known. For example, all or part of the probes or primers may be used to hybridize to the coding sequence. Then, through PCR amplification, the full length sequence may be generated. The full length sequence can be subsequently subcloned into any vector of choice.
  • the primers or probes may be radioactively labeled at the 5' end in order to screen cDNA libraries by conventional means.
  • a primer or probe can be labeled with a radioactive element which provides for an adequate signal as a means for detection and has sufficient half-life to be useful for detection, such as 32p 3j ⁇ , 14c or the like.
  • recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention can be prepared. Many such vectors are illustrated and described above.
  • the preferred nucleic acid vectors are those which are DNA.
  • the preferred recombinant DNA vectors comprise the isolated DNA sequences as given by SEQ ID NO:l or SEQ ID NO:3.
  • the most preferred recombinant DNA vectors comprise the DNA sequence given by SEQ ID NO:3.
  • the type of cloning vector employed depends upon the availability of appropriate restriction sites, the type of host cell in which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., transient expression in an oocyte system, stable transformation as an extrachromosomal element, or integration into the host chromosome), the presence or absence of readily assayable markers (e.g., antibiotic resistance markers, metabolic markers, or the like), and the number of copies of the gene to be present in the cell.
  • readily assayable markers e.g., antibiotic resistance markers, metabolic markers, or the like
  • the type of vector employed to carry the nucleic acids of the present invention may be RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like.
  • a constitutive promoter i.e. a promoter which is functional at all times, instead of a regulatable promoter which may be activated or inactivated by the artisan using heat, addition or removal of a nutrient, addition of an antibiotic, and the like.
  • the practitioner also understands that the amount of the nucleic acid or protein to be produced dictates, in part, the selection of the expression system.
  • Plasmids can be readily modified to construct expression vectors in a variety of organisms, including, but not limited to, E. coli, Sf9 (as host for baculovirus), Spodoptera and Saccharomyces. See: Serook et al., Molecular Cloning: A Laboratory Manual (1989)
  • oligonucleotide-directed site-specific mutagenesis One of the most widely employed techniques for altering a nucleic acid sequence is by way of oligonucleotide-directed site-specific mutagenesis.
  • B Comack "Current Protocols in Molecular Biology", 8.01-8.5.9, (F. Ausubel, et al., eds. 1991).
  • an oligonucleotide whose sequence contains the mutation of interest, is synthesized as described supra.
  • This oligonucleotide is then hybridized to a template containing the wild-type sequence.
  • the template is a single-stranded template.
  • Particularly useful are plasmids which contain regions such as the fl intergenic region. This region allows the generation of single-stranded templates when a helper phage is added to the culture harboring the "phagemid".
  • a DNA-dependent DNA polymerase is then used to synthesize the second strand from the oligonucleotide, complementary to the template DNA.
  • the resulting product is a heteroduplex molecule containing a mismatch due to the mutation in the oligonucleotide.
  • Host cells which harbor the nucleic acids provided by the present invention can also be prepared.
  • One suitable host cell is an Xenopus sp. oocyte which has been injected with RNA or DNA compounds of the present invention.
  • Preferred oocytes of the present invention are those which harbor a sense mRNA of the present invention.
  • Other preferred host cells include HeLa and 293 cells which have been transfected and/or transformed with a vector which comprises a nucleic acid of the present invention.
  • a method for constructing a recombinant host cell capable of expressing SEQ ID NO:2 or SEQ ID NO:4 is also possible with regard to the present invention, said method comprising transforming a host cell with a recombinant DNA vector that comprises an isolated DNA sequence which encodes SEQ ID NO:2 or SEQ ID NO:4.
  • the preferred host cell is 293 cells.
  • a preferred vector for expression is one which comprises SEQ ID NO:l or SEQ ID NO:3, most preferably SEQ ID NO:l.
  • Transformed host cells may be cultured under conditions well known to skilled artisans such that SEQ ID NO: 2 or SEQ ID NO:4 are expressed, thereby producing the sequence of ⁇ - secretase in the recombinant host cell.
  • a further embodiment of the invention consists of a method of isolating and purifying an amyloid precursor protein protease from a host cell expressing said protein.
  • a host cell either prokaryotic or eukaryotic, expressing amyloid precursor protein protease
  • the second step of this embodiment is the isolation of amyloid precursor protein protease from the cultured cells.
  • the cultured cells are lysed by nitrogen cavitation in the presence of protease inhibitors.
  • a soluble fraction is prepared from the lysate by ultracentrifugation.
  • the resulting solution of cytosolic proteins contains ⁇ -secretase and is subjected to a series of purification procedures.
  • the soluble fraction of the cell lysate is run through a series of column chromatography procedures. Anion exchange chromatography is followed by hydrophobic interaction, molecular sizing, and finally another hydrophobic interaction technique where the conditions are such that the ⁇ - secretase binds the resin weakly. Each column is run individually, and the eluate is collected in fractions while monitoring for absorbance at 280 nm. Fractions are assayed for ⁇ -secretase activity, and those fractions with the desired activity are then run over the next column until a homogeneous solution of ⁇ -secretase is obtained.
  • Immunoaffinity purification using anti- ⁇ -secretase antibodies is an alternative to the series of chromatographic procedures already mentioned. Making antiserum or monoclonal antibodies directed against a purified protein is well known in the art, and skilled artisans readily will be able to prepare anti- ⁇ -secretase antibodies. Preparing an immunoaffinity matrix using such antibodies and isolating ⁇ -secretase using the immunoaffinity matrix is also well within the skill of the art. See, AFFINITY CHROMATOGRAPHY PRINCIPLES & METHODS, Pharmacia Fine Chemicals, 1983. The ability of an agent to inhibit the protein of the present invention is essential in the development of a multitude of indications.
  • such an assay includes a method for determining whether a substance is a functional ligand of ⁇ -secretase, said method comprising contacting a functional compound of the ⁇ -secretase with said substance, monitoring enzymatic activity by physically detectable means, and identifying those substances which effect a chosen response.
  • the instant invention provides such a screening system useful for discovering agents which inhibit the ⁇ -secretase, said screening system comprising the steps of:
  • Utilization of the screening system described above provides a sensitive and rapid means to determine compounds which inhibit ⁇ -secretase.
  • This screening system may also be adapted to automated procedures such as a
  • PANDEX® Boxter-Dade Diagnostics
  • a protein protease is prepared as elsewhere described herein, preferably using recombinant DNA technology.
  • a sample of a test compound is then introduced to the reaction vessel containing the protein protease followed by the addition of an appropriate substrate.
  • the substrate may be added simultaneously with the test compound.
  • the desirability of a bioactivity assay system which determines the response of ⁇ -secretase to a compound of interest is clear.
  • the instant invention provides such a bioactivity assay, said assay comprising the steps of: a) transfecting a mammalian host cell with an expression vector comprising DNA encoding ⁇ -secretase;
  • the present invention comprises a method of using said inhibitors of ⁇ -secretase in the treatment of patients with acquired disease states of the brain including Alzheimer's disease.
  • patient refers to a mammal such a rat, mouse, guinea pig, dog, or human. It is understood, however, that the preferred patient for use with the methods herein is a human patient.
  • treatment includes its generally acepted meaning which encompasses prohibiting, preventing, restraining, and slowing, stopping, or reversing the progression, severity, and resulting symptoms of acquired disease states of the brain.
  • themethods of the present invention encompass both therapeutic and prophylactic uses.
  • Compounds which are inhibitors of ⁇ -secretase are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • the present invention also includes methods employing pharmaceutical compositions which contain, as the active ingredient, inhibitors of ⁇ -secretase associated with pharmaceutically acceptable carriers.
  • the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be a solid, semi- solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing for example up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the active compound In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents.
  • compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • the compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the active compound is effective over a wide dosage range.
  • dosages per day normally fall within the range of about 0.5 to about 30 mg/kg of body weight. In the treatment of adult humans, the range of about 1 to about 15 mg/kg/day, in single or divided dose, is especially preferred.
  • the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • Hard gelatin capsules containing the following ingredients are prepared:
  • Quantity Ingredient (mg/capsule)
  • the above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
  • a tablet formula is prepared using the ingredients below:
  • Quantity Ingredient (mg/tablet)
  • Stearic acid 5.0 The components are blended and compressed to form tablets, each weighing 240 mg.
  • a dry powder inhaler formulation is prepared containing the following components:
  • the active mixture is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
  • Tablets each containing 30 mg of active ingredient, are prepared as follows:
  • Quantity Ingredient (mg/tablet)
  • the active ingredient, starch and cellulose are passed through a
  • Capsules each containing 40 mg of medicament are made as follows:
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed, through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.
  • Suppositories each containing 25 mg of active ingredient are made as follows:
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
  • Suspensions each containing 50 mg of medicament per 5.0 ml dose are made as follows:
  • Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg
  • the medicament, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water.
  • the sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
  • Capsules each containing 15 mg of medicament, are made as follows:
  • the active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425 mg quantities.
  • An intravenous formulation may be prepared as follows:
  • a topical formulation may be prepared as follows:
  • the white soft paraffin is heated until molten.
  • the liquid paraffin and emulsifying wax are incorporated and stirred until dissolved.
  • the active ingredient is added and stirring is continued until dispersed.
  • the mixture is then cooled until solid.
  • Sublingual or buccal tablets each containing 10 mg of active ingredient, may be prepared as follows:
  • the glycerol, water, sodium citrate, polyvinyl alcohol, and polyvinylpyrrolidone are admixed together by continuous stirring and maintaining the temperature at about 90°C.
  • the solution is cooled to about 50-55°C and the medicament is slowly admixed.
  • the homogenous mixture is poured into forms made of an inert material to produce a drug-containing diffusion matrix having a thickness of about 2-4 mm. This diffusion matrix is then cut to form individual tablets having the appropriate size.
  • transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
  • transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference.
  • patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • Indirect techniques which are generally preferred, usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs or prodrugs.
  • Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier.
  • the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.
  • the proteins of this invention as well as fragments of these proteins may be used as antigens for the synthesis of antibodies.
  • antibody as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab', Fab2', and Fv fragments), and chimeric, humanized, veneered, resurfaced, or CDR-grafted antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived.
  • the instant invention also encompasses single chain polypeptide binding molecules.
  • antibody as used herein is not limited by the manner in which the antibodies are produced, whether such production is in situ or not.
  • antibody as used in this specification encompasses those antibodies produced by recombinant DNA technology means including, but not limited, to expression in bacteria, yeast, insect cell lines, or mammalian cell lines.
  • mice The production of antibodies, both monoclonal and polyclonal, in animals, especially mice, is well known in the art. See, e.g., C. Milstein, HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, (Blackwell Scientific Pub., 1986); J. Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, (Academic Press, 1983).
  • monoclonal antibodies the basic process begins with injecting a mouse, or other suitable animal, with an immunogen. The mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells, resulting in a hybridoma that reproduces in vitro.
  • the population of hybridomas is screened to isolate individual clones, each of which secretes a single antibody species, specific for the immunogen.
  • the individual antibody species obtained in this way is each the product of a single B cell from the immune animal generated in response to a specific antigenic site, or epitope, recognized on the immunogenic substance.
  • Chimeric antibodies are described in U.S. Patent No. 4,816,567, which issued March 28, 1989 to S. Cabilly, et al. This reference discloses methods and vectors for the preparation of chimeric antibodies. The entire contents of U.S. Patent No. 4,816,567 are herein incorporated by reference.
  • An alternative approach to production of genetically engineered antibodies is provided in U.S. Patent No. 4,816,397, which also issued March 28, 1989 to M.
  • Boss, et al. the entire contents of which are herein incorporated by reference.
  • the Boss patent teaches the simultaneous co-expression of the heavy and light chains of the antibody in the same host cell.
  • the approach of U.S. Patent 4,816,397 has been further refined as taught in European Patent Publication No. 0 239 400, which published September 30, 1987.
  • the teachings of this European patent publication (Winter) are a preferred format for the genetic engineering of the reactive monoclonal antibodies of this invention.
  • the Winter technology involves the replacement of complementarity determining regions (CDRs) of a human antibody with the CDRs of a murine monoclonal antibody thereby converting the specificity of the human antibody to the specificity of the murine antibody which was the source of the CDR regions.
  • This "CDR grafting" technology affords a molecule containing minimal murine sequence and thus is less immunogenic.
  • Single chain antibody technology is yet another variety of genetically engineered antibody which is now well known in the art. See, e.g. R.E. Bird, et al., Science 242:423-426 (1988); PCT Publication No. WO 88/01649, which was published 10 March 1988; United States Patent 5,260,203, issued November 9, 1993, the entire contents of which are herein incorporated by reference.
  • the single chain antibody technology involves joining the binding regions of heavy and light chains with a polypeptide sequence to generate a single polypeptide having the binding specificity of the antibody from which it was derived.
  • the aforementioned genetic engineering approaches provide the skilled artisan with numerous means to generate molecules which retain the binding characteristics of the parental antibody while affording a less immunogenic format.
  • antibodies are used in diagnostics, therapeutics or in diagnostic/therapeutic combinations.
  • diagnosis as used herein is meant testing that is related to either the in vitro or in vivo diagnosis of disease states or biological status in mammals, preferably in humans.
  • therapeutics and “therapeutic/diagnostic combinations” as used herein is respectively meant the treatment or the diagnosis and treatment of disease states or biological status by the in vivo administration to mammals, preferably humans, of the antibodies of the present invention.
  • the antibodies of the present invention are especially preferred in the diagnosis and/or treatment of conditions associated with an excess or deficiency of ⁇ -secretase.

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Abstract

This present invention provides an amyloid precursor protein protease and related nucleic acid compounds thereof. The invention also provides compositions, expression vectors, and transfected host cells as well as assays and methods of use. The compounds of the present invention will further characterize Alzheimer's Disease and other neurodegenerative disease states.

Description

AMYLOID PRECURSOR PROTEIN PROTEASE AND RELATED NUCLEIC ACID COMPOUNDS
This invention relates to proteases which cleave amyloid precursor protein and facilitate β-amyloid peptide release, and accordingly, have utility in identifying compounds which treat or prevent Alzheimer's Disease.
Alzheimer's disease is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately death. Alzheimer's disease is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. Alzheimer's disease has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. -Alzheimer's disease is at present incurable. No treatment that effectively prevents Alzheimer's disease or reverses its symptoms and course is currently known.
The brains of individuals with Alzheimer's disease exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with Alzheimer's disease. Smaller numbers of these lesions in a more restrictive anatomical distribution are also found in the brains of most aged humans who do not have clinical Alzheimer's disease. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D).
The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy), characteristic of Alzheimer's disease and the other disorders mentioned above, is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the β-amyloid peptide (this protein is also referred to in the literature as βAP, Aβ, AβP, A- beta, or β/A4). β-Amyloid peptide was first purified and a partial amino acid sequence was provided by Glenner, et al.. Biochemical and Biophysical Research Communications, 120:885-890 (1984). The isolation procedure and the sequence data for the first 28 amino acids are described in United States Patent 4,666,829, the entire contents of which are herein incorporated by reference.
Several lines of evidence indicate that progressive cerebral deposition of β-amyloid peptide plays a seminal role in the pathogenesis of Alzheimer's disease and can precede cognitive symptoms by years or decades. See, e.g., D. Selkoe, Neuron, 6:487-498 (1991). An important line of evidence is the discovery that missense DNA mutations at amino acid 717 of the 770- amino acid isoform of amyloid precursor protein can be found in affected members, but not unaffected members, of several families with a genetically determined (familial) form of Alzheimer's disease. Goate, et al., Nature (London), 349:704-706 (1990). Genetic linkage studies have demonstrated that this mutation, as well as certain other mutations in the amyloid precursor protein gene, are the specific molecular cause of Alzheimer's disease in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of amyloid precursor protein has been identified as the cause of the β-amyloid peptide deposition disease, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles Alzheimer's disease in some patients. The discovery of these and other mutations in amyloid precursor protein in genetically based cases of Alzheimer's disease support the hypothesis that alteration of amyloid precursor protein and subsequent deposition of its β- amyloid peptide fragment can cause Alzheimer's disease.
Molecular biological and protein chemical analyses have shown that the β-amyloid peptide is a small fragment of a much larger precursor protein, the amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding the amyloid precursor protein has demonstrated that the β-amyloid peptide arises as a peptide fragment that is cleaved from the amyloid precursor protein by proteases.
It is presently believed that a normal (i.e., non-pathogenic) processing of the amyloid precursor protein occurs via cleavage by a putative " -secretase" which cleaves between amino acids 16 and 17 of the β-amyloid peptide region within the protein. Kang, et al., Nature (London), 325:773-776 (1987). It is further believed that pathogenic processing occurs in part via an enzyme designated "β-secretase" which cleaves at the amino-terminus of the β-amyloid peptide region within the precursor protein.
The identification, isolation, and purification of the proteases involved in the processing of amyloid precursor protein would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit formation of β-amyloid peptide.
Despite the progress which has been made in understanding the underlying mechanisms of Alzheimer's disease and other β-amyloid peptide- related diseases, there remains a need to develop methods and compositions for treatment of the diseases. Ideally, the treatment methods would advantageously be based on drugs which are capable of inhibiting β-amyloid peptide release and/or its synthesis in vivo. The present invention provides an isolated protein useful as an amyloid precursor protein protease, said compound comprising the amino acid sequence
Met Leu Arg Arg Arg Gly Ser Pro Gly 1 5
Met Gly Val His Val Gly Ala Ala Leu Gly Ala Leu Trp Phe Cys Leu 10 15 20 25
Thr Gly Ala Leu Glu Val Gin Val Pro Glu Asp Pro Val Val Ala Leu
30 35 40
Val Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly 45 50 55
Phe Ser Leu Ala Gin Leu Asn Leu lie Trp Gin Leu Thr Asp Thr Lys 60 65 70
Gin Leu Val His Ser Phe Ala Glu Gly Gin Asp Gin Gly Ser Ala Tyr 75 80 85
Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala 90 95 100 105
Ser Leu Arg Leu Gin Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr
110 115 120
Cys Phe Val Ser lie Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gin 125 130 135
Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys 140 145 150
Asp Leu Arg Pro Gly Asp Thr Val Thr lie Thr Cys Ser Ser Tyr Gin 155 160 165 Gly Tyr Pro Glu Ala Glu Val Phe Trp Gin Asp Gly Gin Gly Val Pro 170 175 180 185
Leu Thr Gly Asn Val Thr Thr Ser Gin Met Ala Asn Glu Gin Gly Leu 190 195 200
Phe Asp Val His Ser lie Leu Arg Val Val Leu Gly Ala Asn Gly Thr 205 210 215
Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gin Gin Asp Ala His Ser 220 225 230
Ser Val Thr lie Thr Pro Gin Arg Ser Pro Thr Gly Ala Val Glu Val 235 240 245
Gin Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr 250 255 260 265
Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gin Leu 270 275 280
Asn Leu lie Trp Gin Leu Thr Asp Thr Lys Gin Leu Val His Ser Phe 285 290 295
Thr Glu Gly Arg Asp Gin Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu 300 305 310
Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala Ser Leu Arg Leu Gin Arg 315 320 325
Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser lie Arg 330 335 340 345
Asp Phe Gly Ser Ala Ala Val Ser Leu Gin Val Ala Ala Pro Tyr Ser 350 355 360 Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp 365 370 375
Thr Val Thr He Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu 380 385 390
Val Phe Trp Gin Asp Gly Gin Gly Val Pro Leu Thr Gly Asn Val Thr 395 400 405
Thr Ser Gin Met Ala Asn Glu Gin Gly Leu Phe Xaa Xaa Xaa Xaa Xaa 410 415 420 425
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 430 435 440
Xaa Xaa Xaa Xaa Xaa Xaa Asp Ala His Gly Ser Val Thr He Thr Gly 445 450 455
Gin Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu 460 465 470
Ser Val Cys Leu He Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp 475 480 485
Arg Lys He Lys Gin Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp 490 495 500 505
Gin Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gin Pro Leu Lys 510 515 520
His Ser Asp Ser Lys Glu Asp Asp Gly Gin Glu He Ala
525 530 535 hereinafter referred to as SEQ ID NO:2.
More preferred is the amyloid precursor protein protease comprising the amino acid sequence Met Leu Arg Arg Arg Gly Ser Pro Gly 1 5
Met Gly Val His Val Gly Ala Ala Leu Gly Ala Leu Trp Phe Cys Leu 10 15 20 25
Thr Gly Ala Leu Glu Val Gin Val Pro Glu Asp Pro Val Val Ala Leu 30 35 40
Val Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly
45 50 55
Phe Ser Leu Ala Gin Leu Asn Leu He Trp Gin Leu Thr Asp Thr Lys 60 65 70
Gin Leu Val His Ser Phe Ala Glu Gly Gin Asp Gin Gly Ser Ala Tyr 75 80 85
Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala 90 95 100 105
Ser Leu Arg Leu Gin Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr 110 115 120
Cys Phe Val Ser He Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gin 125 130 135
Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys 140 145 150
Asp Leu Arg Pro Gly Asp Thr Val Thr He Thr Cys Ser Ser Tyr Gin 155 160 165
Gly Tyr Pro Glu Ala Glu Val Phe Trp Gin Asp Gly Gin Gly Val Pro 170 175 180 185 Leu Thr Gly Asn Val Thr Thr Ser Gin Met Ala Asn Glu Gin Gly Leu 190 195 200
Phe Asp Val His Ser He Leu Arg Val Val Leu Gly Ala Asn Gly Thr 205 210 215
Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gin Gin Asp Ala His Ser 220 225 230
Ser Val Thr He Thr Pro Gin Arg Ser Pro Thr Gly Ala Val Glu Val 235 240 245
Gin Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr 250 255 260 265
Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gin Leu 270 275 280
Asn Leu He Trp Gin Leu Thr Asp Thr Lys Gin Leu Val His Ser Phe 285 290 295
Thr Glu Gly Arg Asp Gin Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu 300 305 310
Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala Ser Leu Arg Leu Gin Arg 315 320 325
Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser He Arg 330 335 340 345
Asp Phe Gly Ser Ala Ala Val Ser Leu Gin Val Ala Ala Pro Tyr Ser 350 355 360
Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp 365 370 375
Thr Val Thr He Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu 380 385 390
Val Phe Trp Gin Asp Gly Gin Gly Val Pro Leu Thr Gly Asn Val Thr 395 400 405
Thr Ser Gin Met Ala Asn Glu Gin Gly Leu Phe Asp Val His Ser Val 410 415 420 425
Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg 430 435 440
Asn Pro Val Leu Gin Gin Asp Ala His Gly Ser Val Thr He Thr Gly 445 450 455
Gin Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu 460 465 470
Ser Val Cys Leu He Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp 475 480 485
Arg Lys He Lys Gin Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp 490 495 500 505
Gin Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gin Pro Leu Lys 510 515 520
His Ser Asp Ser Lys Glu Asp Asp Gly Gin Glu He Ala
525 530 535 hereinafter referred to as SEQ ID NO:4.
The invention also provides isolated nucleic acid compounds that comprise nucleic acid sequences which encode the amino acid compounds provided. Particularly, the isolated nucleic acid compounds that are provided are preferably DNA, however, nucleic acid compounds which are sense or antisense mRNA are also provided. A particularly preferred nucleic acid compound is the DNA compound comprising the sequence
agctgtcagc cgcctcacag gaag atg ctg cgt egg egg ggc age cct ggc 51 Met Leu Arg Arg Arg Gly Ser Pro Gly
1 5
atg ggt gtg cat gtg ggt gca gcc ctg gga gca ctg tgg ttc tgc etc 99 Met Gly Val His Val Gly Ala Ala Leu Gly Ala Leu Trp Phe Cys Leu 10 15 20 25
aca gga gcc ctg gag gtc cag gtc cct gaa gac cca gtg gtg gca ctg 147 Thr Gly Ala Leu Glu Val Gin Val Pro Glu Asp Pro Val Val Ala Leu 30 35 40
gtg ggc ace gat gcc ace ctg tgc tgc tec ttc tec cct gag cct ggc 195 Val Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly 45 50 55
ttc age ctg gca cag etc aac etc ate tgg cag ctg aca gat ace aaa 243 Phe Ser Leu Ala Gin Leu Asn Leu He Trp Gin Leu Thr Asp Thr Lys 60 65 70
cag ctg gtg cac age ttt get gag ggc cag gac cag ggc age gcc tat 291 Gin Leu Val His Ser Phe Ala Glu Gly Gin Asp Gin Gly Ser Ala Tyr 75 80 85
gcc aac cgc acg gcc etc ttc ccg gac ctg ctg gca cag ggc aac gca 339 Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala 90 95 100 105
tec ctg agg ctg cag cgc gtg cgt gtg gcg gac gag ggc age ttc ace 387 Ser Leu Arg Leu Gin Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr 110 115 120
tgc ttc gtg age ate egg gat ttc ggc age get gcc gtc age ctg cag 435 Cys Phe Val Ser He Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gin 125 130 135
gtg gcc get ccc tac teg aag ccc age atg ace ctg gag ccc aac aag 483 Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys 140 145 150
gac ctg egg cca ggg gac aeg gtg ace ate aeg tgc tec age tac cag 531
Asp Leu Arg Pro Gly Asp Thr Val Thr He Thr Cys Ser Ser Tyr Gin 155 160 165
ggc tac cct gag get gag gtg ttc tgg cag gat ggg cag ggt gtg ccc 579
Gly Tyr Pro Glu Ala Glu Val Phe Trp Gin Asp Gly Gin Gly Val Pro 170 175 180 185
ctg act ggc aac gtg ace aeg teg cag atg gcc aac gag cag ggc ttg 627
Leu Thr Gly Asn Val Thr Thr Ser Gin Met Ala Asn Glu Gin Gly Leu
190 195 200
ttt gat gtg cac age ate ctg egg gtg gtg ctg ggt gca aat ggc ace 675
Phe Asp Val His Ser He Leu Arg Val Val Leu Gly Ala Asn Gly Thr 205 210 215
tac age tgc ctg gtg cgc aac ccc gtg ctg cag cag gat gcg cac age 723 Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gin Gin Asp Ala His Ser 220 225 230
tct gtc ace ate aca ccc cag aga age ccc aca gga gcc gtg gag gtc 771
Ser Val Thr He Thr Pro Gin Arg Ser Pro Thr Gly Ala Val Glu Val 235 240 245
cag gtc cct gag gac ccg gtg gtg gcc eta gtg ggc ace gat gcc ace 819
Gin Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr 250 255 260 265
ctg cgc tgc tec ttc tec ccc gag cct ggc ttc age ctg gca cag etc 867 Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gin Leu 270 275 280
aac etc ate tgg cag ctg aca gac ace aaa cag ctg gtg cac agt ttc 915 Asn Leu He Trp Gin Leu Thr Asp Thr Lys Gin Leu Val His Ser Phe 285 290 295
ace gaa ggc egg gac cag ggc age gcc tat gcc aac cgc aeg gcc etc 963 Thr Glu Gly Arg Asp Gin Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu 300 305 310
ttc ccg gac ctg ctg gca caa ggc aat gca tec ctg agg ctg cag cgc 1011 Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala Ser Leu Arg Leu Gin Arg 315 320 325
gtg cgt gtg gcg gac gag ggc age ttc ace tgc ttc gtg age ate egg 1059 Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser He Arg 330 335 340 345
gat ttc ggc age get gcc gtc age ctg cag gtg gcc get ccc tac teg 1107 Asp Phe Gly Ser Ala Ala Val Ser Leu Gin Val Ala Ala Pro Tyr Ser 350 355 360
aag ccc age atg ace ctg gag ccc aac aag gac ctg egg cca ggg gac 1155 Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp 365 370 375
aeg gtg ace ate aeg tgc tec age tac egg ggc tac cct gag get gag 1203 Thr Val Thr He Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu 380 385 390
gtg ttc tgg cag gat ggg cag ggt gtg ccc ctg act ggc aac gtg ace 1251 Val Phe Trp Gin Asp Gly Gin Gly Val Pro Leu Thr Gly Asn Val Thr 395 400 405
aeg teg cag atg gcc aac gag cag ggc ttg ttt nnn nnn nnn nnn nnn 1299 Thr Ser Gin Met Ala Asn Glu Gin Gly Leu Phe Xaa Xaa Xaa Xaa Xaa 410 415 420 425
nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn 1347 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
430 435 440
nnn nnn nnn nnn nnn nnn gat gcg cac ggc tct gtc ace ate aca ggg 1395 Xaa Xaa Xaa Xaa Xaa Xaa Asp Ala His Gly Ser Val Thr He Thr Gly 445 450 455
cag cct atg aca ttc ccc cca gag gcc ctg tgg gtg ace gtg ggg ctg 1443 Gin Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu 460 465 470
tct gtc tgt etc att gca ctg ctg gtg gcc ctg get ttc gtg tgc tgg 1491 Ser Val Cys Leu He Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp 475 480 485
aga aag ate aaa cag age tgt gag gag gag aat gca gga get gag gac 1539 Arg Lys He Lys Gin Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp 490 495 500 505
cag gat ggg gag gga gaa ggc tec aag aca gcc ctg cag cct ctg aaa 1587 Gin Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gin Pro Leu Lys
510 515 520
cac tct gac age aaa gaa gat gat gga caa gaa ata gcc tga 1629
His Ser Asp Ser Lys Glu Asp Asp Gly Gin Glu He Ala 525 530 535
ccatgaggac cagggagctg ctacccctcc ctacagctcc taccctctgg ctgc 1683
hereinafter referred to as SEQ ID NO: l. However, an even more preferred isolated nucleic acid compound is the DNA compound having the sequence agctgtcagc cgcctcacag gaag atg ctg cgt egg egg ggc age cct ggc 51
Met Leu Arg Arg Arg Gly Ser Pro Gly 1 5
atg ggt gtg cat gtg ggt gca gcc ctg gga gca ctg tgg ttc tgc etc 99
Met Gly Val His Val Gly Ala Ala Leu Gly Ala Leu Trp Phe Cys Leu
10 15 20 25
aca gga gcc ctg gag gtc cag gtc cct gaa gac cca gtg gtg gca ctg 147
Thr Gly Ala Leu Glu Val Gin Val Pro Glu Asp Pro Val Val Ala Leu
30 35 40
gtg ggc ace gat gcc ace ctg tgc tgc tec ttc tec cct gag cct ggc 195 Val Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly
45 50 55
ttc age ctg gca cag etc aac etc ate tgg cag ctg aca gat ace aaa 243
Phe Ser Leu Ala Gin Leu Asn Leu He Trp Gin Leu Thr Asp Thr Lys 60 65 70
cag ctg gtg cac age ttt get gag ggc cag gac cag ggc age gcc tat 291
Gin Leu Val His Ser Phe Ala Glu Gly Gin Asp Gin Gly Ser Ala Tyr 75 80 85
gcc aac cgc aeg gcc etc ttc ccg gac ctg ctg gca cag ggc aac gca 339
Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala 90 95 100 105 tec ctg agg ctg cag cgc gtg cgt gtg gcg gac gag ggc age ttc ace 387
Ser Leu Arg Leu Gin Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr
110 115 120
tgc ttc gtg age ate egg gat ttc ggc age get gcc gtc age ctg cag 435 Cys Phe Val Ser He Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gin 125 130 135 gtg gcc get ccc tac teg aag ccc age atg ace ctg gag ccc aac aag 483 Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys 140 145 150
gac ctg egg cca ggg gac aeg gtg ace ate aeg tgc tec age tac cag 531 Asp Leu Arg Pro Gly Asp Thr Val Thr He Thr Cys Ser Ser Tyr Gin 155 160 165
ggc tac cct gag get gag gtg ttc tgg cag gat ggg cag ggt gtg ccc 579 Gly Tyr Pro Glu Ala Glu Val Phe Trp Gin Asp Gly Gin Gly Val Pro 170 175 180 185
ctg act ggc aac gtg ace aeg teg cag atg gcc aac gag cag ggc ttg 627 Leu Thr Gly Asn Val Thr Thr Ser Gin Met Ala Asn Glu Gin Gly Leu
190 195 200
ttt gat gtg cac age ate ctg egg gtg gtg ctg ggt gca aat ggc ace 675 Phe Asp Val His Ser He Leu Arg Val Val Leu Gly Ala Asn Gly Thr 205 210 215
tac age tgc ctg gtg cgc aac ccc gtg ctg cag cag gat gcg cac age 723 Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gin Gin Asp Ala His Ser 220 225 230
tct gtc ace ate aca ccc cag aga age ccc aca gga gcc gtg gag gtc 771 Ser Val Thr He Thr Pro Gin Arg Ser Pro Thr Gly Ala Val Glu Val 235 240 245
cag gtc cct gag gac ccg gtg gtg gcc eta gtg ggc ace gat gcc ace 819 Gin Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr 250 255 260 265
ctg cgc tgc tec ttc tec ccc gag cct ggc ttc age ctg gca cag etc 867 Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gin Leu
270 275 280 aac etc ate tgg cag ctg aca gac ace aaa cag ctg gtg cac agt ttc 915 Asn Leu He Trp Gin Leu Thr Asp Thr Lys Gin Leu Val His Ser Phe 285 290 295
ace gaa ggc egg gac cag ggc age gcc tat gcc aac cgc aeg gcc etc 963 Thr Glu Gly Arg Asp Gin Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu 300 305 310
ttc ccg gac ctg ctg gca caa ggc aat gca tec ctg agg ctg cag cgc 1011
Phe Pro Asp Leu Leu Ala Gin Gly Asn Ala Ser Leu Arg Leu Gin Arg
315 320 325
gtg cgt gtg gcg gac gag ggc age ttc ace tgc ttc gtg age ate egg 1059 Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser He Arg
330 335 340 345
gat ttc ggc age get gcc gtc age ctg cag gtg gcc get ccc tac teg 1107
Asp Phe Gly Ser Ala Ala Val Ser Leu Gin Val Ala Ala Pro Tyr Ser 350 355 360
aag ccc age atg ace ctg gag ccc aac aag gac ctg egg cca ggg gac 1155
Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp
365 370 375
aeg gtg ace ate aeg tgc tec age tac egg ggc tac cct gag get gag 1203
Thr Val Thr He Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu
380 385 390
gtg ttc tgg cag gat ggg cag ggt gtg ccc ctg act ggc aac gtg ace 1251
Val Phe Trp Gin Asp Gly Gin Gly Val Pro Leu Thr Gly Asn Val Thr
395 400 405
aeg teg cag atg gcc aac gag cag ggc ttg ttt gat gtg cac age gtc 1299 Thr Ser Gin Met Ala Asn Glu Gin Gly Leu Phe Asp Val His Ser Val
410 415 420 425 ctg egg gtg gtg ctg ggt gcg aat ggc ace tac age tgc ctg gtg cgc 1347 Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg 430 435 440
aac ccc gtg ctg cag cag gat gcg cac ggc tct gtc ace ate aca ggg 1395 Asn Pro Val Leu Gin Gin Asp Ala His Gly Ser Val Thr He Thr Gly 445 450 455
cag cct atg aca ttc ccc cca gag gcc ctg tgg gtg ace gtg ggg ctg 1443 Gin Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu 460 465 470
tct gtc tgt etc att gca ctg ctg gtg gcc ctg get ttc gtg tgc tgg 1491 Ser Val Cys Leu He Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp 475 480 485
aga aag ate aaa cag age tgt gag gag gag aat gca gga get gag gac 1539 Arg Lys He Lys Gin Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp 490 495 500 505
cag gat ggg gag gga gaa ggc tec aag aca gcc ctg cag cct ctg aaa 1587 Gin Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gin Pro Leu Lys 510 515 520
cac tct gac age aaa gaa gat gat gga caa gaa ata gcc tga 1629
His Ser Asp Ser Lys Glu Asp Asp Gly Gin Glu He Ala
525 530 535
ccatgaggac cagggagctg ctacccctcc ctacagctcc taccctctgg ctgc 1683
hereinafter referred to as SEQ ID NO:3.
Also provided by the present invention are nucleic acid vectors comprising nucleic acids which encode SEQ ID NO:2 or SEQ ID NO:4 or functional equivalents thereof. The preferred nucleic acid vectors are those which are DNA. Most preferred are DNA vectors which comprise the DNA sequence which is SEQ ID NO:3. Moreover, DNA vectors of the present invention preferably comprise a promoter positioned to drive expression of said DNA sequence. Those vectors wherein said promoter functions in human embryonic kidney cells (293 cells), AV12 cells, yeast cells or E.coli cells are preferred.
The present invention also provides probes and primers useful for molecular biology techniques. A compound which encodes all or part of SEQ ID NO:2 or SEQ ID NO:4 and which is at least 18 consecutive base pairs in length is provided. Preferably, the 18 base pair or more compound is DNA. Most preferred for this use are the DNA compounds which comprise at least 18 consecutive base pairs of SEQ ID NO:l or SEQ ID NO:3.
Host cells which harbor the nucleic acids provided by the present invention are also provided. A preferred host cell is an oocyte. A preferred oocyte is one which has been injected with sense mRNA or DNA compounds of the present invention. A more preferred oocyte is one which has been injected with sense mRNA or DNA compounds of the present invention in conjunction with DNA or sense mRNA which encodes APP.
Further, this invention provides cells into which the nucleic acid compounds of the present invention may be transfected. Host cells include those which are transfected with a nucleic acid compound which encodes SEQ ID NO:2 or SEQ ID NO:4. Preferred cells include host cells transfected with a DNA vector comprising SEQ ID NO:l or SEQ ID NO:3. The preferred transfected host cells which encode SEQ ID NO:2 or SEQ ID NO:4 are 293 cells, AV12 cells, yeast cells and E. coli cells. Also preferred is a host cell which has been co-transfected with a DNA vector which comprises SEQ ID NO:l or SEQ ID NO:3 and a DNA vector which comprises the coding sequence of APP. 293 cells, AV12 cells, yeast cells and E. coli cells are the preferred co-transfected host cells.
Additionally, the invention provides a method for identifying DNA homologous to a probe of the present invention, which comprises combining test nucleic acid with the probe under hybridizing conditions and identifying those test nucleic acids which hybridize.
Assays utilizing the compounds provided by the present invention are also provided. The assays provided determine whether a substance is a ligand for β-secretase, said method comprising contacting β-secretase with said substance, monitoring β-secretase activity by physically detectable means, and identifying those substances which interact with or affect β-secretase. Preferred assays of the present invention include a cell culture assay, a high-performance liquid chromotography (HPLC) assay or a synthetic competition assay. Pre- ferred cell culture assays utilize oocytes, AN12, E. coli, yeast or 293 cells which co-express nucleic acids which encode β-secretase and APP.
The invention also provides methods for constructing a host cell capable of expressing a nucleic acid compound which encodes an amino acid compound comprising SEQ ID ΝO:2 or SEQ ID NO:4, said methods comprising transfecting a host cell with a DNA vector comprising a nucleic acid compound encoding SEQ ID NO:2 or SEQ ID NO:4. A preferred method utilizes 293, AV12, yeast or E. coli cells as the host cells. A more preferred method includes a DNA vector which comprises SEQ ID NO:l or SEQ ID NO:3. A most preferred method includes a DNA vector which comprises SEQ ID NO:3. Another preferred method comprises (a) a DNA vector which comprises SEQ ID NO: 3 and (b) a DNA expression vector which encodes the APP coding sequence.
Additionally, methods for expressing a nucleic acid sequence which encodes SEQ ID NO:2 or SEQ ID NO:4, or functional equivalents thereof , in a transfected host cell, are also provided. These methods comprise culturing a transfected host cell of the present invention under conditions suitable for gene expression. A preferred method utilizes 293, AV12, yeast or E. coli cells as the transfected host cell. A more preferred method utilizes a DNA vector to transfect the host cell. A most preferred method utilizes a DNA vector comprising all or part of SEQ ID NO:3. The present invention also provides processes for isolating and purifying an amyloid precursor protein protease, said process comprising: (a) establishing in a suitable medium, a culture of the host cells transformed with a polynucleotide encoding an amyloid precursor protein protease; and (b) isolating said protease from said culture. Compositions comprising a peptide isolated according to such processes are also provided.
In addition, polyclonal and monoclonal antibodies to the peptides of the present invention are also provided.
The terms and abbreviations used in this document have their normal meanings unless otherwise designated. For example "°C" refers to degrees Celsius; "N" refers to normal or normality; "mmol" refers to millimole or millimoles; "g" refers to gram or grams; "ml" means milliliter or milliliters; "M" refers to molar or molarity; "μg" refers to microgram or micrograms; "μl" refers to microliter or microliters; "pU" refers to pico Unit, and the like. All nucleic acid sequences, unless otherwise designated, are written in the direction from the 5' end to the 3' end, frequently referred to as "5' to 3'".
All amino acid or protein sequences, unless otherwise designated, are written commencing with the amino terminus ("N-terminus") and concluding with the carboxy terminus ("C -terminus").
"Base pair" or "bp" as used herein refers to DNA or RNA. The abbreviations A, C, G, and T correspond to the 5'-monophosphate forms of the deoxyribonucleosides (deoxy)adenosine, (deoxy)cytidine, (deoxy)guanosine, and (deoxy)thymidine, respectively, when they occur in DNA molecules. The abbreviations U, C, G, and A correspond to the 5'-monophosphate forms of the ribonucleosides uridine, cytidine, guanosine, and adenosine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA, heteroduplex base pair may refer to a partnership of A with U or C with G. (See the definition of "complementary", infra.)
The terms "digestion" or "restriction" of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA ("sequence-specific endonucleases"). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can be readily found in the literature.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with a DNA ligase, such as T4 DNA ligase. The term "plasmid" refers to an extrachromosomal (usually) self- replicating genetic element. Plasmids are generally designated by a lower case "p" preceded and/or followed by letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
The term "reading frame" means the nucleotide sequence from which translation occurs "read" in triplets by the translational apparatus of transfer RNA (tRNA) and ribosomes and associated factors, each triplet corresponding to a particular amino acid. To insure against improper translation, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame" being maintained. "Recombinant DNA cloning vector" as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added. The term "recombinant DNA expression vector" as used herein refers to any recombinant DNA cloning vector in which a promoter to control transcription of the inserted DNA has been incorporated.
The term "expression vector system" as used herein refers to a recombinant DNA expression vector in combination with one or more trans- acting factors that specifically influence transcription, stability, or replication of the recombinant DNA expression vector. The trans-acting factor may be expressed from a co-transfected plasmid, virus, or other extrachromosomal element, or may be expressed from a gene integrated within the chromosome.
"Transcription" as used herein refers to the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
The term "transfection" as used herein refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, calcium phosphate co-precipitation, and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
The term "transformation" as used herein means the introduction of DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Methods of transforming bacterial and eukaryotic hosts are well known in the art, many of which methods, such as nuclear injection, protoplast fusion or by calcium treatment using calcium chloride are summarized in J. Sambrook, et al.. MOLECULAR CLONING: A LABORATORY MANUAL, (1989). The term "translation" as used herein refers to the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.
The term "vector" as used herein refers to a nucleic acid compound used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which when combined with appropriate control sequences confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. The term "vector" as used herein includes Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
The terms "complementary" or "complementarity" as used herein refer to the capacity of p urine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules. The following base pairs are related by complementarity: guanine and cytosine; adenine and thymine; and adenine and uracil. As used herein, "complementary" means that the aforementioned relationship applies to substantially all base pairs comprising two single-stranded nucleic acid molecules over the entire length of said molecules. "Partially complementary" refers to the aforementioned relationship in which one of two single-stranded nucleic acid molecules is shorter in length than the other such that a portion of one of the molecules remains single-stranded.
The term "hybridization" as used herein refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. The conditions employed in the hybridization of two completely or nearly completely complementary nucleic acid strands varies with the degree of complementarity of the two strands and the length of the strands. Such techniques and conditions are well known to practitioners in this field. "Isolated amino acid sequence" refers to any amino acid sequence, however constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
"Isolated DNA compound" refers to any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
"Isolated nucleic acid compound" refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location. A "primer" is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation.
The term "promoter" refers to a DNA sequence which directs transcription of DNA to RNA.
A "probe" as used herein is a nucleic acid compound or a fragment thereof which hybridizes with a nucleic acid compound which encodes either the entire sequence of SEQ ID NO:2 or SEQ ID NO:4, a sequence complementary to SEQ ID NO:2 or SEQ ID NO:4, or a part thereof.
The term "EST" or "expressed sequence tag" refers to a fragment or sampling of cDNA which encodes a polypeptide of unknown function. The term "stringency" refers to a set of hybridization conditions which may be varied in order to vary the degree of nucleic acid affinity for other nucleic acid. (See the definition of "hybridization", supra.)
The term "antigenically distinct" as used herein refers to a situation in which antibodies raised against an epitope of the proteins of the present invention, or a fragment thereof, may be used to differentiate between the proteins of the present invention and other β-secretase variants. This term may also be employed in the sense that such antibodies may be used to differentiate between the human β-secretase protein and analogous proteins derived from other species. The term "PCR" as used herein refers to the widely-known polymerase chain reaction employing a thermally-stable polymerase. This technique, as appreciated by those skilled in the art, is employed to amplify a particular nucleic acid fragment. The term "BLAST" as used herein refers to the widely known basic local alignment search tool. This tool consists of a set of computer- based programs designed to permit examination of amino acid and nucleic acid sequence databases for similarity with an isolated sequence of interest. The term "RACE" refers to the widely known rapid amplification of complimentary ends technique, to amplify and obtain the 5' and 3' ends of isolated cDNA.
Skilled artisans will recognize that the proteins of the present invention can be synthesized by a number of different methods. All of the amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis, or recombinant methods. Both methods are described in U.S. Patent 4,617,149, herein incorporated by reference.
The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area. See, e^, H. Dugas and C. Penney, BlOORGANIC CHEMISTRY, (1981) Springer-
Verlag, New York, pgs. 54-92. For examples, peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems. Protected amino acids, such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses. Sequential t-butoxycarbonyl chemistry using double couple protocols are applied to the starting /-.-methyl benzhydryl amine resins for the production of C-terminal carboxamides. For the production of C-terminal acids , the corresponding pyridine-2-aldoxime methiodide resin is used. Asparagine, glutamine, and arginine are coupled using preformed hydroxy benzotriazole esters. The following side chain protection may be used:
Arg, Tosyl
Asp, cyclohexyl Glu, cyclohexyl
Ser, Benzyl
Thr, Benzyl
Tyr, 4-bromo carbobenzoxy
Removal of the t-butoxycarbonyl moiety (deprotection) may be accomplished with trifluoroacetic acid (TFA) in methylene chloride.
Following completion of the synthesis the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees centigrade or below, preferably - 20°C for thirty minutes followed by thirty minutes at 0°C.
After removal of the hydrogen fluoride, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and then lyophilized. Purification is accomplished by size-exclusion chromatography on a Sephadex G-10 (Pharmacia) column in 10% acetic acid. The proteins of the present invention may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired. A general method for the construction of any desired DNA sequence is provided in J. Brown, et al., Methods in Enzymology, 68:109 (1979). See also, J. Sambrook, et al., supra. The basic steps in the recombinant production of desired proteins are:
a) construction of a synthetic or semi-synthetic DNA encoding the protein of interest; b) integrating said DNA into an expression vector in a manner suitable for the expression of the protein of interest, either alone or as a fusion protein;
c) transforming an appropriate eukaryotic or prokaryotic host cell with said expression vector,
d) culturing said transformed or transfected host cell in a manner to express the protein of interest; and
e) recovering and purifying the recombinantly produced protein of interest.
In general, prokaryotes are used for cloning of DNA sequences in constructing the vectors of this invention. Prokaryotes may also be employed in the production of the protein of interest. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins. Other strains of E. coli which may be used (and their relevant genotypes) include the following.
Strain Genotype
DH5a F- (j80dlacZDMl5), D(lacZYA-argF)U169 supE44, l-> hsdR17(rK-, mκ +), recAl, endAl, gyrA96, thi-1, relAl
HB101 supE44, hsdS20(rp ∞B"), recA13, ara-14, proA2 lacYl, galK2, rpsL20, xyl-5, mtl-1, mcrB, mrr JM109 recAl, el4-(mcrA), supE44, endAl, hsdR17(rK ", mκ +), gyrA96, relAl, thi-1, (lac- proAB), F'[traD36, proAB+ ladq,lacZ?M15]
RR1 supE44, hsdS20(rB " mB "), ara-14 proA2, lacYl, galK2, rpsL20, xyl-5, mtl-5
cl776 F-, ton, A53, dapD8, minAl, supE42
(glnN42), D(gal-uvrB)40, minB2, rfb-2, gyrN25, thyA142, oms-2, metC65, oms-1, D(bioH-asd)29, cycB2, cycAl, hsdR2, 1"
294 endA, thr, hsr-, hsm + (U.S. Patent
4,366,246)
LE392 F-, hsdR514 (mr), supE44, supF58, lacYl, or Dlac(I-Y)6, galK2, glaT22, metBl, trpR55, 1-
These strains are all commercially available from suppliers such as: Bethesda Research Laboratories, Gaithersburg, Maryland 20877 and Stratagene Cloning Systems, La Jolla, California 92037; or are readily available to the public from sources such as the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland, 10852-1776. Except where otherwise noted, these bacterial strains can be used interchangeably. The genotypes listed are illustrative of many of the desired characteristics for choosing a bacterial host and are not meant to limit the invention in any way. The genotype designations are in accordance with standard nomenclature. See, for example, J. Sambrook, et al., supra. In addition to the strains of E. coli discussed supra, bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella tvphimurium or Serratia marcescans, and various Pseudomonas species may be used. In addition to these gram-negative bacteria, other bacteria, especially Streptomvces, spp., may be employed in the prokaryotic cloning and expression of the proteins of this invention.
Promoters suitable for use with prokaryotic hosts include the b- lactamase [vector pGX2907 (ATCC 39344) contains the replicon and b- lactamase gene] and lactose promoter systems [Chang et al.. Nature (London), 275:615 (1978); and Goeddel et al.. Nature (London), 281:544
(1979)], alkaline phosphatase, the tryptophan (trp) promoter system [vector pATHl (ATCC 37695) is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter] and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC- 37282). However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine -Dalgarno sequence operably linked to the DNA encoding the desired polypeptides. These examples are illustrative rather than limiting.
The proteins of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the lifespan, increases the yield of the desired peptide, or provides a convenient means of purifying the protein of interest. A variety of peptidases (e.g. trypsin) which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13 in PROTEIN PURIFICATION: FROM MOLECULAR MECHANISMS TO LARGE SCALE PROCESSES, American Chemical Society, Washington, DC (1990).
In addition to cloning and expressing the genes of interest in the prokaryotic systems discussed above, the proteins of the present invention may also be produced in eukaryotic systems. The present invention is not limited to use in a particular eukaryotic host cell. A variety of eukaryotic host cells are available from depositories such as the American Type Culture Collection (ATCC) and are suitable for use with the vectors of the present invention. The choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of the amyloid precursor protein protease-encoding nucleic acids of the present invention. Exemplary host cells suitable for use in the present invention are listed in Table I. Theses exemplary host cells are merely illustrative of the many eukaryotic cells available for use with the present invention and are not meant in any way to limit the scope of the present invention.
Table I
Host Cell Origin Source
HepG-2 Human Liver Hepatoblastoma ATCC HB 8065
CV-1 African Green Monkey Kidney ATCC CCL 70
LLC-MK2 Rhesus Monkey Kidney ATCC CCL 7
3T3 Mouse Embryo Fibroblasts ATCC CCL 92
CHO-K1 Chinese Hamster Ovary ATCC CCL 61
HeLa Human Cervix Epitheloid ATCC CCL 2 RPMI8226 Human Myeloma ATCC CCL 155
H4IIEC3 Rat Hepatoma ATCC CCL 1600
C127I Mouse Fibroblast ATCC CCL 1616
293 Human Embryonal Kidney ATCC CRL 1573
Sf9 Fall armyworm ovary Spodoptera ATCC CRL-1711 frugiperda
HS-Sultan Human Plasma Cell Plasmocytoma ATCC CCL 1484
BHK-21 Baby Hamster Kidney ATCC CCL 10
A preferred cell line employed in the expression of the protein of the present invention is the widely available 293 cell line. As noted, this cell line was constructed from human embryonal kidney tissue and is available from American Type Culture Collection under the accession number ATCC CCL 1573.
A wide variety of vectors, some of which are discussed below, exists for the transformation of mammalian host cells, but the specific vectors described herein are merely illustrative and are in no way intended to limit the scope of the present invention.
Some illustrative vectors include the pSV2-type vectors which comprise segments of the simian virus 40 (SN40) genome that constitute a defined eukaryotic transcription unit-promoter, intervening sequence, and polyadenylation site. In the absence of the SN40 T antigen, the plasmid pSN2-type vectors transform mammalian and other eukaryotic host cells by integrating into the host cell chromosomal DΝA. A large number of plasmid pSV2-type vectors have been constructed, such as plasmid pSN2-gpt, pSN2- neo, pSN2-dhfr, pSN2-hyg, and pSV2-b-globin, in which the SN40 promoter drives transcription of an inserted gene. These vectors are suitable for use with the coding sequences of the present invention and are widely available from sources such as the ATCC or the Northern Regional Research Laboratory (NRRL), 1815 N. University Street, Peoria, Illinois, 61604.
The plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolate reductase (dhfr) gene under the control of the SV40 early promoter. Under the appropriate conditions, the dhfr gene is known to be amplified, or copied, in the host chromosome. This amplification can result in the amplification of closely-associated DNA sequences and can, therefore, be used to increase production of a protein of interest. See, e.g., J. Schimke, Cell, 35:705-713 (1984). Plasmids constructed for expression of the proteins of the present invention in mammalian and other eukaryotic host cells can utilize a wide variety of promoters. The present invention is in no way limited to the use of the particular promoters exemplified herein. Promoters such as the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene, and the major early and late adenovirus genes can be readily isolated and modified to express the genes of the present invention. Eukaryotic promoters can also be used in tandem to drive expression of a coding sequence of this invention. Furthermore, a large number of retroviruses are known that infect a wide range of eukaryotic host cells. The long terminal repeats in the retroviral DNA frequently encode functional promoters and, therefore, may be used to drive expression of the nucleic acids of the present invention. Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention. H. Gorman, et al., Proceedings of the National Academy of Sciences (USA). 79:6777 (1982). The plasmid pMSNi (NRRL B- 15929) comprises the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells. The mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the expression of the nucleic acids of the present invention. The mouse metallothionein promoter is present in the plasmid pdBPN-MMTneo (ATCC 37224) which can serve as the starting material of other plasmids of the present invention.
Another usefull expression vector system employs one of a series of vectors containing the BK enhancer, an enhancer derived from the BK virus, a human papovavirus. The most preferred such vector systems are those which employ not only the BK enhancer but also the adenovirus-2-early region IA (El A) gene product. The E1A gene product (actually, the E1A gene produces two products, which are collectively referred to herein as "the E1A gene product") is an immediate-early gene product of adenovirus, a large DΝA virus. An additional useful eukaryotic expression vector is the phd series of vectors which comprise a BK enhancer in tandem with the adenovirus late promoter to drive expression of useful products in eukaryotic host cells. The construction and method of using the phd plasmid, as well as related plasmids, are described in U.S. Patents 5,242,688, issued September 7, 1993, and 4,992,373, issued February 12, 1991, all of which are herein incorporated by reference. Escherichia coli K12 GM48 cells harboring the plasmid phd are available as part of the permanent stock collection of the Northern Regional Research Laboratory under accession number NRRL B- 18525. The plasmid may be isolated from this culture using standard techniques.
The plasmid phd contains a unique Bell site which may be utilized for the insertion of the gene encoding the protein of interest. The skilled artisan understands that linkers or adapters may be employed in cloning the gene of interest into this Bell site. The phd series of plasmids functions most efficiently when introduced into a host cell which produces the E1A gene product, cell lines such as AN12-664, 293 cells, and others, described supra.
Transformation of the mammalian cells can be performed by any of the known processes including, but not limited to, the protoplast fusion method, the calcium phosphate co-precipitation method, electroporation and the like. See, e.g., J. Sambrook, et al., supra, at 3:16.30-3:16.66.
Other routes of production are well known to skilled artisans. In addition to the plasmid discussed above, it is well known in the art that some viruses are also appropriate vectors. For example, the adenovirus, the adeno- associated virus, the vaccinia virus, the herpes virus, the baculovirus, and the rous sarcoma virus are useful. Such a method is described in U.S. Patent 4,775,624, herein incorporated by reference. Several alternate methods of expression are described in J. Sambrook, et al., supra, at 16.3-17.44.
In addition to prokaryotes and mammalian host cells, eukaryotic microbes such as yeast cultures may also be used. The imperfect fungus Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For expression in Saccharomyces sp., the plasmid YRp7 (ATCC-40053), for example, is commonly used. See, e.g., L. Stinchcomb, et al., Nature (London), 282:39 (1979); J. Kingsman et al.. Gene, 7:141 (1979); S.
Tschemper et al.. Gene, 10:157 (1980). This plasmid already contains the trp_ gene which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [found on plasmid pAP12BD (ATCC 53231) and described in U.S. Patent No. 4,935,350, issued June 19, 1990, herein incorporated by reference] or other glycolytic enzymes such as enolase [found on plasmid p AC 1 (ATCC 39532)], glyceraldehyde-3-phosphate dehydrogenase [derived from plasmid pHcGAPCl (ATCC 57090, 57091)], hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, p ho sp ho glucose isomerase, and glucokinase, as well as the alcohol dehydrogenase and pyruvate decarboxylase genes of Zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991, herein incorporated by reference).
Other yeast promoters, which are inducible promoters, having the additional advantage of their transcription being controllable by varying growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein [contained on plasmid vector
PCL28XhoLHBPV (ATCC 39475) and described in United States Patent No. 4,840,896, herein incorporated by reference], glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose [e.g. GAL1 found on plasmid pRY121 (ATCC 37658)] utilization. Suitable vectors and promoters for use in yeast expression are further described in R.
Hitzeman et al., European Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjunction with the CYC1 promoter on plasmid YEpsec-hllbeta ATCC 67024), also are advantageously used with yeast promoters. Practitioners of this invention realize that, in addition to the above-mentioned expression systems, cloned cDNA may also be employed in the production of transgenic animals in which a test mammal, usually a mouse, in which expression or overexpression of the proteins of the present invention can be assessed. The nucleic acids of the present invention may also be employed in the construction of "knockout" animals in which the expression of the native cognate of the gene is suppressed.
Skilled artisans will recognize that the protease of the present invention, as well as the nucleic acid compounds encoding said protease, can be isolated and purified from cultured host cells transiently expressing β- secretase activity. The following examples further illustrate such isolation and purification processes, however, these examples are not in any way to be interpreted as limiting the scope of the present invention.
Isolation and Purification of β-secretase Example 1
293 Cell Source 293 Cells were obtained as by-products from 25- to 30-day fermentations at Lilly Technology Center. (Eli Lilly and Co., Indianapolis, IN.) Example 2 β-Secretase Activity Assay
The enzymatic activity of beta-secretase was determined by an ELISA assay, modified in form over an assay appearing in application No. PCT/US96/09985, the entire content of which is herein incorporated by reference. Using MBP-APPcl25sw as the substrate, the activity was determined by measuring the MBP-APPc26sw cleavage product with a polyclonal antibody, 192sw, highly specific to the product. MBP-APPcl25sw is a fusion substrate in which maltose binding protein is fused to the 125- residue C-terminal portion of the Swedish amyloid precursor protein. For the enzymatic activity analysis, one picounit (pU) is defined as the amount of the enzyme that catalyzes the conversion of MBP-APP125sw to one picomole of MBP-APPc26sw per ml in 2-hr under the optimized reaction conditions of the Elisa assay (see Table II, infra). To calculate the enzyme specific activity, the protein concentration was first determined using the well known Bio-Rad microassay method. (Rio-Rad Laboratories, Hercules, CA, 94547) Example 3 β-Secretase Purification
The purification of beta-secretase was carried out by a protocol modified over the purification methods appearing in application No. PCT/US96/09985, the entire content of which is herein incorporated by reference. The enzyme purification protocol was undertaken at 4 C. β- Secretase activity and protein recovery data are provided in Table III, infra.
Step 1:
Membrane Extract: About 400-g of 293 cells were resuspended in 2,000-ml of 20 mM HEPES (Sigma Chemical Co., St Louis, MO), pH 7.5, 2 mM EDTA (Sigma Chemical Co., St Louis, MO) and 0.25% sucrose (Buffer IA). The cell suspension was homogenized in an 1000-ml aliquot and the cells were broken with an Brinkmann Homogenizer (POLYTRON, Switzerland) according to the following conditions: at setting 4 for 2- and 1-min; and at setting 5 for 1- and 0.5-min (with 3-min cooling time after each homogenization). The broken cell suspension was centrifuged at l,000xg for 20-min; the resulting supernatant (post-nuclear supernatant; PNSl) was saved. The pellet was resuspended in 1,200-ml of Buffer IA, homogenized , broken and centrifuged as described above; the supernatant (PNS2) was saved and the pellet was discarded. PNSl and PNS2 were combined (2,700-ml) and centrifuged at 45,000xg for 1-hr; the resulting pellet (P2; containing membranes) was saved and the supernatant was discarded. This pellet was resuspended in 3,200-ml of 20 mM MES (Sigma Chemical Co., St Louis, MO), pH 6.0, 2 mM EDTA, 0.5% Triton X-100, 150 mM NaCl, 0.2 mM AEBSF, 5 mg/ml leupeptin, 2 mg/ml E64 and 1 mg/ml pepstatin A (Buffer IB); all four protease inhibitors were from Calbiochem; San Diego, CA). The suspension was stirred continuously for 1-hr for extraction of beta-secretase and centrifuged at 16,000xg for 1-hr; the pellet was discarded. The resulting supernatant (3,120- ml) was adjusted to pH 7.5 with 1 M Tris base and filtered through 0.45-mm Zapcap-S (Schleicher & Schuell; Keene, NH); the filtrate was the P2 membrane extract (P2ME, 3,170-ml).
A second P2ME was prepared similarly from another 400g of 293 cells. The combined beta-secretase activity and protein data for the P2ME fractions are shown in Table III. However, beta-secretase activity from each P2ME was purified separately in the next step.
Step 2:
Wheat Germ Agglutinin (WGA) Agarose Eluate: The first P2ME was loaded to a WGA-Agarose column (Vector Lab.; Burlingame, CA) (1000-ml), pre-equilibrated with 1500-ml of 20 mM Tris, pH 7.5, 2 mM EDTA, 0.5% Triton X-100 and 150 mM NaCl (Buffer 2A). The WGA column, which binds to N-acetyl-D-glucosamine glycopeptides, was washed with 1000-ml of Buffer 2A and bound proteins were eluted with a linear gradient of 0-10% chitin hydrolysate (Vector Lab.) in a total volume of 900-ml constructed from Buffer 2A and Buffer 2B (20 mM Tris, pH 7.5, 2 mM EDTA, 0.5% Triton X-100 and 10% chitin hydrolysate). The fractions containing higher specific activities of beta-secretase were pooled as the WGA-eluate (180-ml).
The WGA-Agarose column was regenerated and equilibrated. The second WGA-eluate (170-ml) was obtained similarly from the second P2ME. The two WGA-eluates were combined. (see Table III). In addition, the two WGA- Agarose washings were combined. (see Table III). The WGA-eluate and the WGA-wash were purified together in the next step. Step 3:
HiTrap Q (HiQ) Eluate: The WGA-eluate and the WGA-wash were diluted 6x and 4x, respectively, with 20 mM Tris, pH 8.0, 2 mM EDTA and 0.2% Triton X-100 (Buffer 3A) to reduce Triton X-100 and NaCl concentrations. Both diluted enzyme preparations were loaded consecutively to four connected, fast flow anion exchanger HiTrap Q columns (4x5-ml; Amersham Pharmacia Biotech, Piscataway, NJ), pre-equilibrated with 100-ml of Buffer 3A, and the column was washed with 200-ml of Buffer 3A. Bound proteins were eluted first with a linear gradient of 0-500 mM NaCl in buffer 3A in a total volume of 420-ml and then eluted with 100-ml of Buffer 3B (Buffer 3A plus 500 mM NaCl). The fractions containing higher specific activities were pooled as the HiQ-eluate. (Table III) The HiQ-eluate was used directly in the next step.
Step 4:
Hydroxylapatite (HA) Wash: The HiQ-eluate was loaded to a Hydroxylapatite column (10-ml; Bio-Rad, Hercules, CA), pre-equilibrated with 100-ml of Buffer 4A (same as Buffer 3A), the column flow-through was collected. HA separation of proteins is based on charge absorption and desorption. The column was washed with 50-ml of Buffer 4A, and the bound proteins were eluted from the column with a linear gradient of 0-500 mM potassium phosphate in Buffer 4A in a total volume of 100-ml. Among the three fractions (the flow-through, the wash, and the eluate), the HA-wash (50-ml) showed the highest total , and specific activity of beta-secretase (Table III) and, thus, was used in the next step.
Step 5:
Mono Q Eluate. The HA-wash was de-salted to about 15 mM NaCl with Buffer 5A (same as Buffer 4A) using Centriprep 50 (Millipore, Bedford, MA ). The de-salted HA-wash was loaded to a high resolving strong anion exchanger Mono Q column (1-ml; Amersham Pharmacia Biotech), pre- equilibrated with 30-ml of Buffer 5A. The column was washed with 30-ml of buffer 5A, and bound proteins were eluted first with a linear gradient of 0- 500 mM in Buffer 5A in a total volume of 20-ml and then eluted with 10-ml of Buffer 5B (Buffer 5A plus 500 mM NaCl). Two activity peaks were observed and the active fractions from each activity peak were pooled. (Table III) The two Mono Q-eluates (2-ml & 4-ml)were purified separately in the next step.
Step 6:
Superdex (S) 200 Eluate. Mono Q-eluate 1 was loaded to a high resolution gel-filtration Superdex 200 column (300-ml; Amersham Pharmacia Biotech), pre-equilibrated with 900-ml of 20 mM Tris, pH 8.0, 2 mM EDTA, 0.2% Triton X-100, 1 mM MgCl2, 1 mM CaCl2 and 150 mM NaCl (Buffer 6). The proteins were eluted with 300-ml of Buffer 6. Two activity peaks were observed and the active fractions from both peaks were pooled together (S- 200-eluate 1, 19.2-ml)(Table III) and used in the next step. Mono Q-eluate 2 was similarly purified by the same Superdex 200 column; three activity peaks were observed. The two Superdex 200-eluates that showed the higher specific activities (S-200 eluates 2a & 2b; Table III) were used separately in the next step.
Step 7:
DEAE Eluate. S-200-eluates 1 and 2a were de-salted with a Sepharose G-25 column (100-ml; Amersham Pharmacia Biotech) and S-200-eluate 2b was diluted 6x with Buffer 7A (same as 5A) to 25 mM NaCl.. To the resulting S- 200-eluates; 200-, 200- and 100 μl of DEAE-Sepharose was added, respectively. For effective binding of the activity to the resin, the three samples were agitated continuously overnight. The samples were centrifuged at 10,000xg for 10-min to collect DEAE-Sepharose beads. The bound proteins were eluted from DEAE-Sepharose beads of the three samples with, respectively, 2x200-, 2x400- and 2x100 μl of Buffer 7B (Buffer 7A plus 1 M NaCl). The three DEAE-eluates (400-, 800-, and 200 μl; Table III) were treated separately in the next step.
Step 8:
De-Gylcosylation. To each of the three DEAE-eluates, beta- mercaptoethanol (reducing agent) was added to a final concentration of 3 mM. PNGase F (100 mU/40-ml from Glyko, Novato, CA); an N-linked de- glycosylation enzyme, was added as three aliquots to each sample to a final ratio of about 1:4 (V/V). After each PNGase addition, the samples were placed in a 37 °C incubator for one day. After a total incubation time of three days, beta-secretase activity was stable in two samples and there was a slight (about 20%) activity loss in the other sample(Table III). The three samples(De-Gly) were purified separately in the next and last step.
Step 9:
Mini Q Eluate. Each of the three De-Gly samples was de-salted with a
Sephadex PD-10 column (Amersham Pharmacia Biotech). Desalted De-Gly sample 1 was loaded to a Mini Q PE column (0.8-ml from Amersham
Pharmacia Biotech), a high resolving anion exchanger, pre-equilibrated with Buffer 9 A (same as Buffer 7A). The bound proteins were washed and eluted with a step-wise NaCl gradient in Buffer 7A in a total volume of 35.6-ml as shown below: 7.6-ml at 0 mM NaCl, 16-ml from 0 to 250 mM, 8-ml from 250 to 500 mM and 4-ml at 500 mM. Two activity peaks were observed. Desalted De-Gly 2a were purified in a similar way; two activity peaks were also observed. Desalted De-Gly 2b sample was purified with a Mini Q PC column (0.24-ml from Amersham Pharmacia Biotech) using a step-wise NaCl gradient in Buffer 7A in a total volume of 13.2-ml (4.8-ml at 0 mM NaCl, 4.8- ml from 0 to 250 mM, 2.4-ml from 250 to 500 mM and 1.2-ml at 500 mM). Only one major activity peak was observed. The resulting beta-secretase activity and protein data are shown in Table III.
Beta-secretase activity was purified up to 3, 000-fold to apparent homogeneity by the 9-step procedure. Note, however, that the actual activity enrichment was likely much greater excluding Triton X-100 interference to the low level protein determination in Step 9 above.
Table II Optimized Assay for β-Secretase Activity
Assay Parameter Optimized Activity Conditions
Enzyme* 0.015-0.095 pU for Linear Response (With 0.02 to 0.06 % Triton X-100)
Substrate -60 nM for Maximal Activity (With -20 mM Guanidine HC1)
pH 5.25 to 5.75 for Maximal Activity
Reaction Time > 4 hours for Linear Response
Kinetic Constants
Km 300 nM max 2 nmol/hr/mg protein
0.313-μl of Mini Q-Eluate 2a .
Note: The enzymatic reaction was carried out at room temperature. The purified enzyme was stable at 4 or -20 C for at least two weeks. Table III
Fraction (step) Total Activity Total Protein Sp. Act. Recovery (pU) (mg) (pU/mg) (%)
P2ME (1) 35,038 23,245 1.507 100
WGA-Wash (2) 2,160 206 10.49 6
WGA-E* (2) 8,330 652 12.78 24
HiQ-E* (3) 5,998 259 23.16 17
HA-Wash (4) 2,550 20.4 125.0 7.3
Mono Q-E*l (5) 1,248 3.99 312.7 3.6
Mono Q-E*2 (5) 2,240 6.43 348.4 6.4
S-200-E*l (6) 199.5 0.90 221.7 0.6
S-200-E*2b (6) 386.5 1.54 251.0 1.1
S-200-E*2c (6) 60.0 0.16 375.0 0.2
DEAE-E*1 (7) 239.3 0.406 574.5 0.7
DEAE-E*2 (7) 771.8 0.642 1202 2.2
DEAE-E*3 (7) 8.35 0.048 174.0
De-Gly* 1 (8) 222.5 0.397 560.5 0.6
De-Gly*2 (8) 534.3 0.634 842.7 1.5
De-Gly*3 (8) 11.33 0.046 246.2
Mmι Q-E*la (9) 121.8 0.049 2485 0.3
Mmι Q-E*lb (9) 76.8 0.045 1706 0.2
Mini Q-E*2a (9) 247.0** 0.087 2839 0.7
Mini Q-E*2b (9) 369.8** 0.121 3056 1.1
Mini Q-E*3 (9) 2.95 0.009 327.8 *E = Eluate; FT = Flow-Through; De-Gly = De-Glycosylated
** Mini Q-E la and Mini Q-E lb each contained two activity peaks.
Mini Q-Eluate 2a represents the first peak from fraction Q-E la combined with the first peak from fraction
Q-E lb.
Mini Q-Eluate 2b represents the second peak from fraction Q-E la combined with the second peak from fraction Q-E lb.
Skilled artisans will recognize that some alterations of SEQ ID NO: 2 or SEQ ID NO: 4 will fail to change the function of the amino acid compound. For instance, some hydrophobic amino acids may be exchanged for other hydrophobic amino acids. Those altered amino acid compounds which confer substantially the same function in substantially the same manner as the exemplified amino acid compounds are also encompassed within the present invention. Typical such conservative substitutions attempt to preserve the: (a) secondary or tertiary structure of the polypeptide backbone; (b) the charge or hydrophobicity of the residue; or (c) the bulk of the side chain. Some examples of such conservative substitutions of amino acids, resulting in the production of proteins which are functional equivalents of the protein of SEQ ID NO:2 or SEQ ID NO:4 are shown in Table II, infra.
Table IV
Original Residue Exemplary Substitutions
_ Ser, Gly
Arg Lys Asn Gin, His
Asp Glu
Cys Ser
Gin Asn
Glu Asp Gly Pro, Ala
His Asn, Gin lie Leu, Val
Leu He, Val
Lys Arg, Gin, Glu Mel Leu, lie
Phe Met, Leu, Gyr
Ser Thr
Thr Ser
Trp Tyr Tyr Trp, Phe Val He, Leu
These substitutions may be introduced into the protein in a variety of ways, such as during the chemical synthesis or by chemical modification of an amino acid side chain after the protein has been prepared.
Alterations of the protein having a sequence which corresponds to the sequence of SEQ ID NO:2 or SEQ ID NO:4 may also be induced by alterations of the nucleic acid compounds which encodes these proteins. These mutations of the nucleic acid compound may be generated by either random mutagenesis techniques, such as those techniques employing chemical mutagens, or by site-specific mutagenesis employing oligonucleotides. In addition, allelic variants of the gene encoding the protein of the present invention may also be purified by the processes of the present invention. Those nucleic acid compounds which confer substantially the same function in substantially the same manner as the exemplified nucleic acid compounds are also encompassed within the present invention.
Other embodiments of the present invention include nucleic acid compounds which comprise isolated nucleic acid sequences which encode SEQ ID NO:2 or SEQ ID NO:4. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one nucleic acid triplet due to the degeneracy of the amino acid code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.
The full length nucleic acid clones which encode the amino acid sequences given by SEQ ID NO:2 and SEQ ID NO:4, where determined using standard methods, essentially as follows. Fractions of purified beta- secretase, as isolated by the methods described in Examples 1 through 3 above, were applied to an SDS-polyacrylamide gel. Following electrophoresis, the proteins were transferred electrophoretically to a polyvinyldiene difluoride (PVDF) membrane and stained with coomassie blue. The stained protein bands were then excised from the membrane and placed in an automatic protein sequencer where the amino acid sequence of the p-rotein was determined using widely known automated Edman chemistry. N- terminal fragments of the determined protein sequence, of approximately 17 —20 amino acids, were then searched against protein sequence databases using commercially available BLAST methodology. A BLAST analysis of the proprietary INCYTE database (INCYTE Pharmaceuticals, Palo Alto, CA, 94304) revealed a series of ESTs showing homology to the determined experimental sequence. The ESTs were subsequently assembled into a contiguous cDNA sequence.
The transcript of the assembled ESTs, however, did not contain a starting methionine. In order to elongate the 5' end of the experimental sequence 5' RACE (rapid amplification of complimentary ends) was performed, [see generally: RACE, as described in M.A. Fohrman, PCR Protocols: A Guide to Methods and Applications, Mclnnis et al., Eds., Academic Press, Inc, San Diego, CA, pp. 28-29 (1990); see also U.S. Patent No. 5,470,722; and also see Ausubel et al, Current Protocols in Molecular Biology, Ch. 15, Eds., Wiley and Sons, NT. (1989-1990)] After designing suitable primers based on the information contained in the EST contiguous cDNA sequence, RACE-ready cDNA (CLONTECH Laboratories, Palo Alto, CA, 94303) was used as a template to elongate and amplify the 5' end until a starting methionine was detected. PCR was then employed to generate the full length cDNA clone. E. coli were subsequently transformed with the full length cDNAs comprising SEQ ID NO:l and SEQ ID NO:3 in order to generate sufficient copies of the nucleic acids for sequence determination.
The gene encoding the amyloid precursor protein protease of the present invention may also be produced using synthetic methodology, which synthesis is well known in the art. See, e.g., E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana, Methods in Enzvmology. 68:109-151 (1979). Additionally, the nucleic acid sequences corresponding to the gene encoding the amyloid precursor protein protease of the present invention, can also be generated using conventional DNA synthesizing apparatuses such as the Applied Biosystems Model 380A or 380B DNA synthesizers, (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) These synthesizers employ phosphoramidite chemistry. In the alternative, the more traditional phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. See, e.g., M.J. Gait, ed., OLIGONUCLEOTIDE SYNTHESIS, A PRACTICAL APPROACH, (1984).
The synthetic amyloid precursor protein protease gene may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into expression and amplification plasmids. The restriction sites are chosen so as to properly orient the coding sequence of the target enzyme with control sequences to achieve proper in-frame reading and expression of the amyloid precursor protein protease molecule. A variety of other such cleavage sites may be incorporated depending on the particular plasmid constructs employed and may be generated by techniques well known in the art.
In an alternative methodology, the desired DNA sequences can be generated using the polymerase chain reaction as described in U.S. Patent No. 4,889,818, which is herein incorporated by reference. In addition to the deoxyribonucleic acid compounds described supra, the present invention also encompasses ribonucleic acid compounds which have a sequence which encodes SEQ ID NO:2 or SEQ ID NO:4. Particularly, the present invention provides the RNA sequence represented by
GCUGUCAGC CGCCUCACAG GAAGAUGCUG CGUCGGCGGG GCAGCCCUGG 50
CAUGGGUGUG CAUGUGGGUG CAGCCCUGGG AGCACUGUGG UUCUGCCUCA 100
CAGGAGCCCU GGAGGUCCAG GUCCCUGAAG ACCCAGUGGU GGCACUGGUG 150
GGCACCGAUG CCACCCUGUG CUGCUCCUUC UCCCCUGAGC CUGGCUUCAG 200
CCUGGCACAG CUCAACCUCA UCUGGCAGCU GACAGAUACC AAACAGCUGG 250
UGCACAGCUU UGCUGAGGGC CAGGACCAGG GCAGCGCCUA UGCCAACCGC 300
ACGGCCCUCU UCCCGGACCU GCUGGCACAG GGCAACGCAU CCCUGAGGCU 350 GCAGCGCGUG CGUGUGGCGG ACGAGGGCAG CUUCACCUGC UUCGUGAGCA 400
UCCGGGAUUU CGGCAGCGCU GCCGUCAGCC UGCAGGUGGC CGCUCCCUAC 450
UCGAAGCCCA GCAUGACCCU GGAGCCCAAC AAGGACCUGC GGCCAGGGGA 500
CACGGUGACC AUCACGUGCU CCAGCUACCA GGGCUACCCU GAGGCUGAGG 550
UGUUCUGGCA GGAUGGGCAG GGUGUGCCCC UGACUGGCAA CGUGACCACG 600
UCGCAGAUGG CCAACGAGCA GGGCUUGUUU GAUGUGCACA GCAUCCUGCG 650
GGUGGUGCUG GGUGCAAAUG GCACCUACAG CUGCCUGGUG CGCAACCCCG 700
UGCUGCAGCA GGAUGCGCAC AGCUCUGUCA CCAUCACACC CCAGAGAAGC 750
CCCACAGGAG CCGUGGAGGU CCAGGUCCCU GAGGACCCGG UGGUGGCCCU 800
AGUGGGCACC GAUGCCACCC UGCGCUGCUC CUUCUCCCCC GAGCCUGGCU 850
UCAGCCUGGC ACAGCUCAAC CUCAUCUGGC AGCUGACAGA CACCAAACAG 900
CUGGUGCACA GUUUCACCGA AGGCCGGGAC CAGGGCAGCG CCUAUGCCAA 1000
CCGCACGGCC CUCUUCCCGG ACCUGCUGGC ACAAGGCAAU GCAUCCCUGA 1050
GGCUGCAGCG CGUGCGUGUG GCGGACGAGG GCAGCUUCAC CUGCUUCGUG 1100
AGCAUCCGGG AUUUCGGCAG CGCUGCCGUC AGCCUGCAGG UGGCCGCUCC 1150
CUACUCGAAG CCCAGCAUGA CCCUGGAGCC CAACAAGGAC CUGCGGCCAG 1200
GGGACACGGU GACCAUCACG UGCUCCAGCU ACCGGGGCUA CCCUGAGGCU 1250
GAGGUGUUCU GGCAGGAUGG GCAGGGUGUG CCCCUGACUG GCAACGUGAC 1300
CACGUCGCAG AUGGCCAACG AGCAGGGCUU GUUUGAUGUG CACAGCGUCC 1350 UGCGGGUGGU GCUGGGUGCG AAUGGCACCU ACAGCUGCCU GGUGCGCAAC 1400
CCCGUGCUGC AGCAGGAUGC GCACGGCUCU GUCACCAUCA CAGGGCAGCC 1450
UAUGACAUUC CCCCCAGAGG CCCUGUGGGU GACCGUGGGG CUGUCUGUCU 1500
GUCUCAUUGC ACUGCUGGUG GCCCUGGCUU UCGUGUGCUG GAGAAAGAUC 1550
AAACAGAGCU GUGAGGAGGA GAAUGCAGGA GCUGAGGACC AGGAUGGGGA 1560
GGGAGAAGGC UCCAAGACAG CCCUGCAGCC UCUGAAACAC UCUGACAGCA 1600
AAGAAGAUGA UGGACAAGAA AUAGCCUGAC CAUGAGGACC AGGGAGCUGC 1650
UACCCCUCCC UACAGCUCCU ACCCUCUGGC UGC 1683
which is herein referred to as SEQ ID NO:5.
The ribonucleic acids of the present invention may be prepared using the polynucleotide synthetic methods discussed supra or they may be prepared enzymatically using any one of various RNA polymerases to transcribe a DNA template. This invention also provides nucleic acids, RNA or DNA, which are complementary to SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5
The present invention also provides probes and primers useful for molecular biology techniques. A compound which encodes for SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5; or a nucleic acid complimentary to SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5; or a fragment thereof and which is at least 18 base pairs in length, and which will selectively hybridize to genomic DNA or messenger RNA encoding a β-secretase protease, is provided. Preferably, the 18 or more base pair compound is DNA.
Primers and probes may be obtained by means well known in the art. For example, once a protein of interest is isolated, restriction enzymes and subsequent gel separation may be used to isolate the fragment of choice. The term "selectively hybridize" as used herein may refer to either of two situations. In the first such embodiment of this invention, the nucleic acid compounds described supra hybridize to DNA or RNA encoding a human β-secretase protease under more stringent hybridization conditions than these same nucleic acid compounds would hybridize to nucleic acids encoding analogous β-secretases of another species. In the second such embodiment of this invention, these probes hybridize to the DNA or RNA encoding the β-secretase under more stringent hybridization conditions than other related compounds, including nucleic acid sequences encoding other amyloid precursor protein proteases.
These probes and primers can be prepared enzymatically as described supra. In a most preferred embodiment, however, these probes and primers are synthesized using chemical means as described herein.
Those skilled in the art will recognize the techniques associated with probes and primers are well known. For example, all or part of the probes or primers may be used to hybridize to the coding sequence. Then, through PCR amplification, the full length sequence may be generated. The full length sequence can be subsequently subcloned into any vector of choice. Alternatively, the primers or probes may be radioactively labeled at the 5' end in order to screen cDNA libraries by conventional means. A primer or probe can be labeled with a radioactive element which provides for an adequate signal as a means for detection and has sufficient half-life to be useful for detection, such as 32p 3jτ, 14c or the like. Other materials which can be used to label the primer or probe include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and chemiluminescent compounds. An appropriate label can be selected having regard to the rate of hybridization and binding of the primer or probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. As discussed supra, recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention can be prepared. Many such vectors are illustrated and described above. The preferred nucleic acid vectors are those which are DNA. The preferred recombinant DNA vectors comprise the isolated DNA sequences as given by SEQ ID NO:l or SEQ ID NO:3. The most preferred recombinant DNA vectors comprise the DNA sequence given by SEQ ID NO:3.
The skilled artisan understands that the type of cloning vector employed depends upon the availability of appropriate restriction sites, the type of host cell in which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., transient expression in an oocyte system, stable transformation as an extrachromosomal element, or integration into the host chromosome), the presence or absence of readily assayable markers (e.g., antibiotic resistance markers, metabolic markers, or the like), and the number of copies of the gene to be present in the cell. The type of vector employed to carry the nucleic acids of the present invention may be RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. When preparing an expression vector, the skilled artisan understands that there are many variables to be considered. One such example is the use of a constitutive promoter, i.e. a promoter which is functional at all times, instead of a regulatable promoter which may be activated or inactivated by the artisan using heat, addition or removal of a nutrient, addition of an antibiotic, and the like. The practitioner also understands that the amount of the nucleic acid or protein to be produced dictates, in part, the selection of the expression system. For experiments examining the amount of the protein expressed on the cell membrane or for experiments examining the biological function of an expressed membrane protein, for example, it may be unwise to employ an expression system which produces too much of the protein. The addition or subtraction of certain sequences, such as a signal sequence preceding the coding sequence, may be employed by the practitioner to influence localization of the resulting polypeptide. Such sequences added to or removed from the nucleic acid compounds of the present invention are encompassed within this invention.
Plasmids can be readily modified to construct expression vectors in a variety of organisms, including, but not limited to, E. coli, Sf9 (as host for baculovirus), Spodoptera and Saccharomyces. See: Serook et al., Molecular Cloning: A Laboratory Manual (1989)
One of the most widely employed techniques for altering a nucleic acid sequence is by way of oligonucleotide-directed site-specific mutagenesis. B Comack, "Current Protocols in Molecular Biology", 8.01-8.5.9, (F. Ausubel, et al., eds. 1991). In this technique an oligonucleotide, whose sequence contains the mutation of interest, is synthesized as described supra. This oligonucleotide is then hybridized to a template containing the wild-type sequence. In a most preferred embodiment of this technique, the template is a single-stranded template. Particularly useful are plasmids which contain regions such as the fl intergenic region. This region allows the generation of single-stranded templates when a helper phage is added to the culture harboring the "phagemid".
After the annealing of the oligonucleotide, to the template, a DNA-dependent DNA polymerase is then used to synthesize the second strand from the oligonucleotide, complementary to the template DNA. The resulting product is a heteroduplex molecule containing a mismatch due to the mutation in the oligonucleotide. After DNA replication by the host cell a mixture of two types of plasmid are present, the wild-type and the newly constructed mutant. This technique permits the introduction of convenient restriction sites such that the coding sequence may be placed immediately adjacent to whichever transcriptional or translational regulatory elements are employed by the practitioner.
The construction protocols utilized for E. coli can be followed to construct analogous vectors for other organisms, merely by substituting, if necessary, the appropriate regulatory elements using techniques well know to skilled artisans.
Host cells which harbor the nucleic acids provided by the present invention can also be prepared. One suitable host cell is an Xenopus sp. oocyte which has been injected with RNA or DNA compounds of the present invention. Preferred oocytes of the present invention are those which harbor a sense mRNA of the present invention. Other preferred host cells include HeLa and 293 cells which have been transfected and/or transformed with a vector which comprises a nucleic acid of the present invention.
A method for constructing a recombinant host cell capable of expressing SEQ ID NO:2 or SEQ ID NO:4 is also possible with regard to the present invention, said method comprising transforming a host cell with a recombinant DNA vector that comprises an isolated DNA sequence which encodes SEQ ID NO:2 or SEQ ID NO:4. The preferred host cell is 293 cells. A preferred vector for expression is one which comprises SEQ ID NO:l or SEQ ID NO:3, most preferably SEQ ID NO:l. Transformed host cells may be cultured under conditions well known to skilled artisans such that SEQ ID NO: 2 or SEQ ID NO:4 are expressed, thereby producing the sequence of β- secretase in the recombinant host cell.
A further embodiment of the invention consists of a method of isolating and purifying an amyloid precursor protein protease from a host cell expressing said protein. In this embodiment, a host cell, either prokaryotic or eukaryotic, expressing amyloid precursor protein protease, is cultured in an appropriate medium until a substantial cell mass has been obtained. The second step of this embodiment is the isolation of amyloid precursor protein protease from the cultured cells. Two methods for purifying amyloid precursor protein protease from a non-transformed mammalian cell line are described in United States Patent Number 5,328,842, the entire contents of which are herein incorporated by reference. The following summarizes those methods.
Once grown and harvested, the cultured cells are lysed by nitrogen cavitation in the presence of protease inhibitors. A soluble fraction is prepared from the lysate by ultracentrifugation. The resulting solution of cytosolic proteins contains β-secretase and is subjected to a series of purification procedures.
The soluble fraction of the cell lysate is run through a series of column chromatography procedures. Anion exchange chromatography is followed by hydrophobic interaction, molecular sizing, and finally another hydrophobic interaction technique where the conditions are such that the β- secretase binds the resin weakly. Each column is run individually, and the eluate is collected in fractions while monitoring for absorbance at 280 nm. Fractions are assayed for β-secretase activity, and those fractions with the desired activity are then run over the next column until a homogeneous solution of β-secretase is obtained.
Immunoaffinity purification using anti- β-secretase antibodies is an alternative to the series of chromatographic procedures already mentioned. Making antiserum or monoclonal antibodies directed against a purified protein is well known in the art, and skilled artisans readily will be able to prepare anti-β-secretase antibodies. Preparing an immunoaffinity matrix using such antibodies and isolating β-secretase using the immunoaffinity matrix is also well within the skill of the art. See, AFFINITY CHROMATOGRAPHY PRINCIPLES & METHODS, Pharmacia Fine Chemicals, 1983. The ability of an agent to inhibit the protein of the present invention is essential in the development of a multitude of indications. In developing agents which act as inhibitors of β-secretase, it would be desirable, therefore, to determine those agents which interact with the protein of the present invention. Generally, such an assay includes a method for determining whether a substance is a functional ligand of β-secretase, said method comprising contacting a functional compound of the β-secretase with said substance, monitoring enzymatic activity by physically detectable means, and identifying those substances which effect a chosen response.
The instant invention provides such a screening system useful for discovering agents which inhibit the β-secretase, said screening system comprising the steps of:
a) isolating a β-secretase;
b) exposing said β-secretase to a potential inhibitor of the β- secretase;
c) introducing a suitable substrate;
d) quantifying the amount of cleavage of the substrate relative to a control in which no potential inhibitor has been introduced.
This allows one to rapidly screen for inhibitors of β-secretase. Utilization of the screening system described above provides a sensitive and rapid means to determine compounds which inhibit β-secretase. This screening system may also be adapted to automated procedures such as a
PANDEX® (Baxter-Dade Diagnostics) system allowing for efficient high- volume screening of potential therapeutic agents. In such a screening protocol a protein protease is prepared as elsewhere described herein, preferably using recombinant DNA technology. A sample of a test compound is then introduced to the reaction vessel containing the protein protease followed by the addition of an appropriate substrate. In the alternative the substrate may be added simultaneously with the test compound. The desirability of a bioactivity assay system which determines the response of β-secretase to a compound of interest is clear. The instant invention provides such a bioactivity assay, said assay comprising the steps of: a) transfecting a mammalian host cell with an expression vector comprising DNA encoding β-secretase;
b) culturing said host cell under conditions such that the β- secretase protein is expressed;
c) exposing said host cell so transfected to a test compound; and
d) measuring the change in a physiological condition known to be influenced by β-secretase relative to a control in which the transfected host cell is not exposed to a test compound.
The present invention comprises a method of using said inhibitors of β-secretase in the treatment of patients with acquired disease states of the brain including Alzheimer's disease. As used herein, "patient" refers to a mammal such a rat, mouse, guinea pig, dog, or human. It is understood, however, that the preferred patient for use with the methods herein is a human patient. As used herein, the term "treatment" (or "treat" or "treating") includes its generally acepted meaning which encompasses prohibiting, preventing, restraining, and slowing, stopping, or reversing the progression, severity, and resulting symptoms of acquired disease states of the brain. As such,themethods of the present invention encompass both therapeutic and prophylactic uses. Compounds which are inhibitors of β-secretase are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
The present invention also includes methods employing pharmaceutical compositions which contain, as the active ingredient, inhibitors of β-secretase associated with pharmaceutically acceptable carriers. In making the compositions of the present invention the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi- solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing for example up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. The compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
The active compound is effective over a wide dosage range. For examples, dosages per day normally fall within the range of about 0.5 to about 30 mg/kg of body weight. In the treatment of adult humans, the range of about 1 to about 15 mg/kg/day, in single or divided dose, is especially preferred. However, it will be understood that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day.
For preparing solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.
The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
The following examples illustrate the pharmaceutical compositions of the present invention.
Formulation Preparation 1
Hard gelatin capsules containing the following ingredients are prepared:
Quantity Ingredient (mg/capsule)
Active Ingredient 30.0
Starch 305.0
Magnesium stearate 5.0
The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
Formulation Preparation 2
A tablet formula is prepared using the ingredients below:
Quantity Ingredient (mg/tablet)
Active Ingredient 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0 The components are blended and compressed to form tablets, each weighing 240 mg.
Formulation Preparation 3
A dry powder inhaler formulation is prepared containing the following components:
Ingredient Weight % Active Ingredient 5
Lactose 95
The active mixture is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
Formulation Preparation 4
Tablets, each containing 30 mg of active ingredient, are prepared as follows:
Quantity Ingredient (mg/tablet)
Active Ingredient 30.0 mg
Starch 45.0 mg
Microcrystalline cellulose 35.0 mg
Polyvinylpyrrolidone (as 10% solution in water) 4.0 mg Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1.0 mg
Total 120 mg
The active ingredient, starch and cellulose are passed through a
No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at
50-60°C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg.
Formulation Preparation 5
Capsules, each containing 40 mg of medicament are made as follows:
Quantity
Ingredient (mg/capsule)
Active Ingredient 40.0 mg
Starch 109.0 mg Magnesium stearate 1.0 mg
Total 150.0 mg
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed, through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.
Formulation Preparation 6
Suppositories, each containing 25 mg of active ingredient are made as follows:
Ingredient Amount Active Ingredient 25 mg
Saturated fatty acid glycerides to 2,000 mg
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.
Formulation Preparation 7
Suspensions, each containing 50 mg of medicament per 5.0 ml dose are made as follows:
Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose (11%)
Microcrystalline cellulose (89%) 50.0 mg
Sucrose 1.75 g
Sodium benzoate 10.0 mg
Flavor and Color q.v.
Purified water to 5.0 ml
The medicament, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
Formulation Preparation 8
Capsules, each containing 15 mg of medicament, are made as follows:
Quantity
Ingredient (mg/capsule)
Active Ingredient 15.0 mg Starch 407.0 mg
Magnesium stearate 3.0 mg
Total 425.0 mg
The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425 mg quantities.
Formulation Preparation 9
An intravenous formulation may be prepared as follows:
Ingredient Quantity
Active Ingredient 250.0 mg
Isotonic saline 1000 ml
Formulation Preparation 10
A topical formulation may be prepared as follows:
Ingredient Quantity
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g White Soft Paraffin to 100 g
The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid.
Formulation Preparation 11
Sublingual or buccal tablets, each containing 10 mg of active ingredient, may be prepared as follows:
Quantity Ingredient Per Tablet
Active Ingredient(s) 10.0 mg
Glycerol 210.5 mg
Water 143.0 mg
Sodium Citrate 4.5 mg
Polyvinyl Alcohol 26.5 mg
Polyvinylpyrrolidone 15.5 mg Total 410.0 mg
The glycerol, water, sodium citrate, polyvinyl alcohol, and polyvinylpyrrolidone are admixed together by continuous stirring and maintaining the temperature at about 90°C. When the polymers have gone into solution, the solution is cooled to about 50-55°C and the medicament is slowly admixed. The homogenous mixture is poured into forms made of an inert material to produce a drug-containing diffusion matrix having a thickness of about 2-4 mm. This diffusion matrix is then cut to form individual tablets having the appropriate size.
Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
Frequently, it will be desirable or necessary to introduce the pharmaceutical composition to the brain, either directly or indirectly. Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier. One such implantable delivery system, used for the transport of biological factors to specific anatomical regions of the body, is described in U.S. Patent 5,011,472, issued April 30, 1991, which is herein incorporated by reference.
Indirect techniques, which are generally preferred, usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs or prodrugs.
Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier. Alternatively, the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.
The proteins of this invention as well as fragments of these proteins may be used as antigens for the synthesis of antibodies. The term "antibody" as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab', Fab2', and Fv fragments), and chimeric, humanized, veneered, resurfaced, or CDR-grafted antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived. The instant invention also encompasses single chain polypeptide binding molecules.
The term "antibody" as used herein is not limited by the manner in which the antibodies are produced, whether such production is in situ or not. The term "antibody" as used in this specification encompasses those antibodies produced by recombinant DNA technology means including, but not limited, to expression in bacteria, yeast, insect cell lines, or mammalian cell lines.
The production of antibodies, both monoclonal and polyclonal, in animals, especially mice, is well known in the art. See, e.g., C. Milstein, HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, (Blackwell Scientific Pub., 1986); J. Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, (Academic Press, 1983). For the production of monoclonal antibodies the basic process begins with injecting a mouse, or other suitable animal, with an immunogen. The mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells, resulting in a hybridoma that reproduces in vitro. The population of hybridomas is screened to isolate individual clones, each of which secretes a single antibody species, specific for the immunogen. The individual antibody species obtained in this way is each the product of a single B cell from the immune animal generated in response to a specific antigenic site, or epitope, recognized on the immunogenic substance. Chimeric antibodies are described in U.S. Patent No. 4,816,567, which issued March 28, 1989 to S. Cabilly, et al. This reference discloses methods and vectors for the preparation of chimeric antibodies. The entire contents of U.S. Patent No. 4,816,567 are herein incorporated by reference. An alternative approach to production of genetically engineered antibodies is provided in U.S. Patent No. 4,816,397, which also issued March 28, 1989 to M. Boss, et al., the entire contents of which are herein incorporated by reference. The Boss patent teaches the simultaneous co-expression of the heavy and light chains of the antibody in the same host cell. The approach of U.S. Patent 4,816,397 has been further refined as taught in European Patent Publication No. 0 239 400, which published September 30, 1987. The teachings of this European patent publication (Winter) are a preferred format for the genetic engineering of the reactive monoclonal antibodies of this invention. The Winter technology involves the replacement of complementarity determining regions (CDRs) of a human antibody with the CDRs of a murine monoclonal antibody thereby converting the specificity of the human antibody to the specificity of the murine antibody which was the source of the CDR regions. This "CDR grafting" technology affords a molecule containing minimal murine sequence and thus is less immunogenic.
Single chain antibody technology is yet another variety of genetically engineered antibody which is now well known in the art. See, e.g. R.E. Bird, et al., Science 242:423-426 (1988); PCT Publication No. WO 88/01649, which was published 10 March 1988; United States Patent 5,260,203, issued November 9, 1993, the entire contents of which are herein incorporated by reference. The single chain antibody technology involves joining the binding regions of heavy and light chains with a polypeptide sequence to generate a single polypeptide having the binding specificity of the antibody from which it was derived. The aforementioned genetic engineering approaches provide the skilled artisan with numerous means to generate molecules which retain the binding characteristics of the parental antibody while affording a less immunogenic format. These antibodies are used in diagnostics, therapeutics or in diagnostic/therapeutic combinations. By "diagnostics" as used herein is meant testing that is related to either the in vitro or in vivo diagnosis of disease states or biological status in mammals, preferably in humans. By "therapeutics" and "therapeutic/diagnostic combinations" as used herein is respectively meant the treatment or the diagnosis and treatment of disease states or biological status by the in vivo administration to mammals, preferably humans, of the antibodies of the present invention. The antibodies of the present invention are especially preferred in the diagnosis and/or treatment of conditions associated with an excess or deficiency of β-secretase.

Claims

WHAT IS CLAIMED IS:
1. An isolated amino acid compound functional as an amyloid precursor protein protease which comprises the amino acid sequence given by SEQ ID NO:2 or SEQ ID NO:4, or a functional equivalent thereof, or a fragment of at least 6 continuous amino acids thereof.
2. A nucleic acid compound encoding the amino acid compound of Claim 1.
3. A nucleic acid compound encoding the amino acid compound of Claim 1 which comprises the nucleic acid sequence given by SEQ ID NO:l or SEQ ID NO:3.
4. A composition comprising an isolated nucleic acid compound containing a sequence encoding an amyloid precursor protein protease as claimed in Claim 2, wherein said sequence is selected from the group consisting of
(a) SEQ ID NO:l or SEQ ID NO:3;
(b) nucleotides 25 through 1629 of SEQ ID NO:l or SEQ ID NO: 3;
(c) SEQ ID NO:5;
(d) nucleotides 25 through 1629 of SEQ ID NO: 5;
(e) a nucleic acid complimentary to (a), (b), (c), or (d); and (f) a fragment of (a), (b), (c), (d), or (e) that is at least 18 bases in length and which will selectively hybridize to human genomic DNA encoding an amyloid precursor protein protease.
5. The composition as claimed in Claim 4 wherein the isolated nucleic acid is deoxyribonucleic acid.
6. The composition as claimed in Claim 5 wherein the deoxyribonucleic acid is SEQ ID NO:l or SEQ ID NO: 3, or a sequence complimentary to SEQ ID NO:l or SEQ ID NO:3.
7. The composition of Claim 5 wherein the deoxyribonucleic acid is nucleotides 25 through 1629 of SEQ ID NO:l or SEQ ID NO:3, or a sequence complimentary to nucleotides 25 through 1629 of SEQ ID NO:l.
8. The composition as claimed in Claim 4 wherein the isolated nucleic acid is ribonucleic acid.
9. The composition as claimed in Claim 8 wherein the ribonucleic acid is SEQ ID NO:5 or a sequence complimentary to SEQ ID NO:5.
10. The composition of Claim 8 wherein the ribonucleic acid is nucleotides 25 through 1629 of SEQ ID NO:5 or a sequence complimentary to nucleotides 25 through 1629 of SEQ ID NO:5.
11. An expression vector capable of producing an amyloid precursor protein protease in a host cell which comprises a nucleic acid compound as claimed in Claim 4 in combination with regulatory elements necessary for expresison of the nucleic acid compound in a host cell.
12. An expression vector as claimed in Claim 11 for use in a host cell wherein said host cell is a mammalian cell.
13. An expression vector as claimed in Claim 11 for use in a host cell wherein said host cell is a prokaryotic cell.
14. A transfected host cell harboring an expression vector as claimed in Claim 11.
15. A transfected host cell as claimed in Claim 14 wherein the host cell is a mammalian cell.
16. A transfected host cell as claimed in Claim 14 wherein the host cell is a prokaryotic cell.
17. A method to identify compounds which inhibit the synthesis or release of beta-amyloid protien, which method comprises:
(a) isolating a β-secretase;
(b) exposing said β-secretase to a potential inhibitor of the β- secretase;
(c) introducing a suitable substrate;
(d) quantifying the amount of cleavage of the substrate relative to a control in which no potential inhibitor has been introduced.
18. The method of Claim 17 wherein said β-secretase comprises the amino acid sequence given by SEQ ID NO:2 or SEQ ID NO:4.
19. A method of evaluating the effectiveness of a test compound for the treatment or prevention of a condition associated with the synthesis or release of beta-amyloid protein, which method comprises:
(a) transfecting a mammalian host cell with an expression vector comprising DNA encoding β-secretase;
(b) culturing said host cell under conditions such that the β- secretase protein is expressed;
(c) exposing said host cell so transfected to a test compound; and (d) measuring the change in a physiological condition known to be influenced by β-secretase relative to a control in which the transfected host cell is not exposed to a test compound.
20. The method of Claim 19 wherein said β-secretase comprises the amino acid sequence given by SEQ ID NO:2 or SEQ ID NO:4.
21. A method of treating or preventing Alzheimer's Disease or other neurodegenerative disorders in a patient which comprises administering to a patient in need thereof, an effective amount of an inhibitor of the amyloid precursor protein protease given by the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
22. A pharmaceutical composition comprising an inhibitor of the amyloid precursor protein protease given by SEQ ID NO:2 or SEQ ID NO:4, in combination with one or more pharmaceutically acceptable excipients, carriers, or diluents therefor.
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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001018204A1 (en) * 1999-09-03 2001-03-15 Curagen Corporation Polynucleotides encoding members of the human b lymphocyte activation antigen b7 family and polypeptides encoded thereby
WO2002022683A1 (en) * 2000-09-12 2002-03-21 Kirin Beer Kabushiki Kaisha Novel dendritic cell wall membrane and use thereof
US6420534B1 (en) 1998-09-24 2002-07-16 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses thereof
US6627739B1 (en) 1999-02-10 2003-09-30 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2003014293A3 (en) * 2001-08-02 2003-12-24 Lilly Co Eli Novel polypeptide analogs and fusions and their methods of use
EP1212344A4 (en) * 1999-09-03 2004-08-04 Human Genome Sciences Inc POLYNUCLEOTIDES, POLYPEPTIDES AND ANTIBODIES TYPE B7
US6835565B1 (en) 1998-09-24 2004-12-28 Pharmacia & Upjohn Company Alzheimer's disease secretase
US6844148B1 (en) 1998-09-24 2005-01-18 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6965018B2 (en) 2000-06-06 2005-11-15 Bristol-Myers Squibb Company Antibodies directed to B7-related polypeptide, BSL-2
WO2005119262A3 (en) * 2004-05-27 2006-06-15 Galapagos Nv Methods, compositions and compound assays for inhibiting amyloid-beta protein production
US7115410B1 (en) 1999-02-10 2006-10-03 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7368554B2 (en) 2000-06-06 2008-05-06 Bristol-Myers Squibb Company Polynucleotides encoding BSL2v1c2-Ig
US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7527969B2 (en) 2002-06-19 2009-05-05 Raven Biotechnologies, Inc. RAAG10 cell surface target and a family of antibodies recognizing that target
US7718774B2 (en) 2006-11-08 2010-05-18 Macrogenics, Inc. TES7 and antibodies that bind thereto
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US8394815B2 (en) 2002-08-19 2013-03-12 Lorus Therapeutics Inc. 2,4,5-trisubstituted imidazoles and their use as anti-microbial agents
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US8969372B2 (en) 2003-11-14 2015-03-03 Aptose Boisciences Inc. Aryl imidazoles and their use as anti-cancer agents
US9150656B2 (en) 2010-03-04 2015-10-06 Macrogenics, Inc. Antibodies reactive with B7-H3, immunologically active fragments thereof and uses thereof
US9309247B2 (en) 2013-03-20 2016-04-12 Lorus Therapeutics Inc. 2-substituted imidazo[4,5-D]phenanthroline derivatives and their use in the treatment of cancer
US9441049B2 (en) 2010-03-04 2016-09-13 Macrogenics, Inc. Antibodies reactive with B7-H3 and uses thereof
US9487587B2 (en) 2013-03-05 2016-11-08 Macrogenics, Inc. Bispecific molecules that are immunoreactive with immune effector cells of a companion animal that express an activating receptor and cells that express B7-H3 and uses thereof
US10961311B2 (en) 2016-04-15 2021-03-30 Macrogenics, Inc. B7-H3 binding molecules, antibody drug conjugates thereof and methods of use thereof
US11104957B2 (en) 2013-10-04 2021-08-31 Aptose Biosciences, Inc. Compositions and methods for treating cancers
US11149047B2 (en) 2017-10-30 2021-10-19 Aptose Biosciences, Inc. Aryl imidazoles for treatment of cancer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0653154A2 (en) * 1993-11-12 1995-05-17 Hoechst Japan Limited Transgenic animal for alzheimer's disease
WO1996040885A2 (en) * 1995-06-07 1996-12-19 Athena Neurosciences, Inc. β-SECRETASE, ANTIBODIES TO β-SECRETASE, AND ASSAYS FOR DETECTING β-SECRETASE INHIBITION
WO1999046281A2 (en) * 1998-03-10 1999-09-16 Genentech, Inc. Novel polypeptides and nucleic acids encoding the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0653154A2 (en) * 1993-11-12 1995-05-17 Hoechst Japan Limited Transgenic animal for alzheimer's disease
WO1996040885A2 (en) * 1995-06-07 1996-12-19 Athena Neurosciences, Inc. β-SECRETASE, ANTIBODIES TO β-SECRETASE, AND ASSAYS FOR DETECTING β-SECRETASE INHIBITION
WO1999046281A2 (en) * 1998-03-10 1999-09-16 Genentech, Inc. Novel polypeptides and nucleic acids encoding the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENEMBL 6 June 1996 (1996-06-06), HILLIER ET AL.: "yx480g8.r1 soares melanocyte 2NbHM Homo sapiens cDNA clone IMAGE:265022 5' similar to contains Alu repetitive element", XP002148339 *
DATABASE SWISSPROT 1 June 1998 (1998-06-01), HUSAINI ET AL.: "Bos taurus butyrophilin fragment", XP002148340 *

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