Assays using amyloid precursor proteins with modified beta-secretase cleavage sites to monitor beta-secretase activity

a technology of beta-secretase and amyloid precursor proteins, which is applied in the field of alzheimer's disease, can solve the problems of no approved pharmaceuticals for treatment and inconvenient identification of assays, and achieve the effect of improving the substrate of -secretas

Inactive Publication Date: 2007-09-20
MERCK SHARP & DOHME CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this assay is not suitable for identifying substances, such as inhibitors of β- or γ-secretase, that would prevent the formation of Aβ.
Despite some progress in identifying β-secretase inhibitors, there are currently no approved pharmaceuticals for the treatment or prevention of Alzheimer's disease that are believed to exert their therapeutic effect through the inhibition of β-secretase.

Method used

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  • Assays using amyloid precursor proteins with modified beta-secretase cleavage sites to monitor beta-secretase activity
  • Assays using amyloid precursor proteins with modified beta-secretase cleavage sites to monitor beta-secretase activity
  • Assays using amyloid precursor proteins with modified beta-secretase cleavage sites to monitor beta-secretase activity

Examples

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example 1

[0344] Polynucleotide sequences that code the mutants disclosed herein, and compared in Table 1 (shown in Example 4, below), were constructed as follows. All mutant polynucleotide constructs were derived from a plasmid designated pRBR899 or pRBR121. Both plasmids carry βAPP695-derived cDNA (either wild type or with the K612V mutation) operatively linked to the CMV promoter. The plasmid pRBR121 was generated by site directed mutagenesis via PCR with an annealed oligonucleotide in which the codon for the K at position 612 (based on the 695 amino acid version of APP) was replaced by a codon for V.

[0345] Thereafter aliquots of both pRBR899 and pRBR121 separately underwent replacement of the 26 bp BglII-EcoRI fragment of βAPP695 with an oligonucleotide that had been prepared to introduce a specific desired substitution of one or more of the four amino acids at the four positions flanking the β-secretase cleavage site (resulting in desired mutations of the APP in one or more of the four ...

example 2

[0348] Antibodies of the present invention were raised against and recognize epitopes (alternately referred to as “neo-epitopes”) formed by the β-secretase cleavage of the modified β-secretase substrates of the present invention. Antibodies were raised against polypeptides mimicking the P1 free carboxylic acid end of a modified sAPPβ that is exposed following cleavage by β-secretase of the modified β-secretase substrates. Antibodies also were raised against polypeptides mimicking the P1′-free amino group end of a modified carboxyterminal fragment (CTFβ) that is exposed following cleavage by β-secretase of the modified β-secretase substrates. At a minimum the modification of modified sAPPβ and the modified CTFβ comprises modification of the amino acid sequences at the P2-P1-P1′-P2′β-secretase cleavage site. An example of an antibody raised to the P1 free carboxylic acid end of a modified sAPPβ, and an example of an antibody raised to the P1′-free amino group end of a modified carboxy...

example 3

[0370] The following provides an example, not meant to be limiting, of a cell-based assay that uses modified APP substrate and measures rate of enzymatic break-down of such substrate by detection of a secreted fragment (sAPpβ) of such modified APP substrate.

[0371] A stable cell line has been generated by transfecting APP695 containing the HA-myc-FLAG epitope in the amino portion of the protein, the K612V (APP695 numbering) mutation altering the alpha-cleavage site, and the modified NFEV BACE cleavage site into HEK293T cells. Standard cell biology protocols were used to generate the cell line. (Ref: Jakoby, W. B. and I. H. Pastan, Eds. (1979). Cell Culture. Methods in Enzymology. San Diego, Calif., Academic Press, Inc.) A polyclonal antibody that recognizes the COOH-terminus of the BACE cleavage product of the NFEV-variant of APP has also been generated. This antibody, called 2081 / anti-NF IgG, was generated against the SEVNF peptide and recognizes the cleaved NF-COOH terminus genera...

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Abstract

Provided are methods of identifying inhibitors of β-secretase that employ modified β-secretase substrates. The modified β-secretase substrates have β-secretase cleavage sites that are altered from wild type. The amino acid sequences of the altered β-secretase cleavage sites contain different amino acids in at least one of the positions P2-P1-P1′-P2′ of the β-secretase cleavage site. Many of the modified β-secretase substrates are more efficient substrates for β-secretase than are corresponding substrates having wild-type sequences, that is, these modified substrates are more susceptible to enzymatic breakdown by β-secretase. Recombinant polynucleotide molecules encoding the modified β-secretase substrates are provided. Antibodies that recognize cleavage products of the modified β-secretase substrates are provided. Stable cell lines expressing the modified β-secretase substrates are provided. Transgenic animals expressing the modified β-secretase substrates are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation in part of, and claims benefit of priority under 35 U.S.C. §120, 363, and 365(c) of International Application No. PCT / US02 / 15590, filed May 17, 2002, which claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application, No. 60 / 316,115, filed Aug. 30, 2001, and of U.S. Provisional Application, No. 60 / 292,591, filed Aug. 30, 2001. These applications, and all references cited therein, are incorporated by reference into this application.STATEMENT REGARDING FEDERALLY SPONSORED R&D [0002] Not applicable. REFERENCE TO MICROFICHE APPENDIX [0003] Not applicable. FIELD OF THE INVENTION [0004] The present invention is directed to the field of Alzheimer's disease. In particular, the present invention provides novel methods of identifying substances that are specific inhibitors of the cleavage of amyloid precursor protein by β-secretase where the methods employ novel polypeptides, namely, modif...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/00C07K14/00A61K38/00C07K5/107C07K5/11C07K5/113C07K7/06C07K14/47C07K16/18C12P21/06C12Q1/37G01N33/50G01N33/68
CPCA01K2217/05G01N2800/52A61K38/00C07K5/1016C07K5/1019C07K5/1021C07K7/06C07K14/4711C07K16/18C12P21/06C12Q1/37G01N33/5088G01N33/6896G01N2500/00A01K2267/0306
Inventor HAZUDA, DARIA JEANCHEN DODSON, ELIZABETHLAI, MING-TAINXU, MINSHI, XIAO-PINGSIMON, ADAM J.WU, GUOXINLI, YUEMINGREGISTER, BRUCE
Owner MERCK SHARP & DOHME CORP
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