Patent AU-2004241151-A1 (AU)
Title: Method for screening inhibitors of the gamma-secretase
Inventor: LEBERER EKKEHARD
IPC: C12N15/62
Filed: 2004-04-29
Abstract: Detecting (M1) activity of gamma -secretase, involves incorporating transgene encoding a fusion protein and comprising first nucleotide sequence (FNS), second nucleotide sequence (SNS), and a promoter, into a cell and expressing fusion protein, forming a first and second partial protein by cleaving the fusion protein using gamma -secretase present in cell; and detecting the first and/or second partial protein. Detecting (M1) the activity of gamma -secretase, involves: (a) obtaining a transgene that encode a fusion protein comprising a first nucleotide sequence (FNS) which codes for a protein having the amino acid sequence of Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Trp-Ile-Ala-Thr-Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu (S1), a second nucleotide sequence (SNS) which codes for a signal peptide, at the 5' end of FNS, a promoter, and if appropriate further coding and/or non-coding nucleotide sequence; (b) incorporating the transgene into a cell and expressing the fusion protein; (c) forming a first partial protein having amino acid sequence of Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val, and a second partial protein having amino acid sequence of Val-Ile-Val-Ile-Thr-Leu-Val-Met-Leu, by cleaving the fusion protein within (S1) using gamma -secretase present in the cell; and (d) detecting the first and/or second partial protein, or determining the amount of second partial protein and determining the activity of gamma -secretase using the determined amount of second partial protein, where the fusion protein contains (S1) and further does not contain one or more peptides acting as a signal for endo- or exocytosis and/or protease cleavage site. Independent claims are also included for: (1) a transgene (I) comprising FNS, SNS at the 5' end of FNS, promoter, and at least one further nucleotide sequence that codes for a DNA-binding domain and for a transcription-activating domain, at the 3' end of FNS; (2) a vector (V1) comprising (I); (3) a cell (II) comprising (I); (4) a transgenic C. elegans (III), comprising (I); (5) a yeast cell (IV) comprising (I); (6) a cell (V) comprising (I), a cDNA library and a reporter plasmid; (7) identifying (M2) substances that inhibit the activity of gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase, involves: (a) producing transgenic non-human organism or a transgenic cell, expressing and containing reporter plasmid carrying a protein binding site, a minimal promoter and a reporter gene, transgene comprising FNS, SNS, promoter and/or further non-coding and/or coding nucleotide sequence, and if appropriate, a cDNA which encodes gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase, incubating the transgenic non-human organism or the transgenic cell with a test substance, and determining the amount of second partial protein; (b) incorporating the transgene encoding the fusion protein having a signal peptide and (S1), reporter plasmid, and if appropriate, a cDNA which encodes gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase, into a cell, determining the expression of the fusion protein encoded by the transgene and a cDNA which encodes gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase encoded by the cDNA, in presence of the test substance, and determining the amount of second partial protein, where when the fusion protein is cleaved by the gamma -secretase present in the cell, the first and second partial proteins are formed, and when the fusion protein is not cleaved by the gamma -secretase present in the cell, the first and second partial proteins are not formed; or (c) determining the expression of the transgene encoding a fusion protein having a signal peptide and (S1), in the presence of a test substance, and determining the effect of the test substance based on the amount of the second partial protein formed; (8) inhibitor (VI) of gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase, identified by (M2); (9) a pharmaceutical composition (PC) comprising a pharmaceutical active compound inhibiting activity of gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase, identified by (M2); and (10) kit for detecting the activity of gamma -secretase, subunit protein of gamma -secretase or gamma -secretase-like proteinase, comprising (I), V1 or any one of (II)-(V). ACTIVITY : Neuroprotective; Nootropic. No biological data given. MECHANISM OF ACTION : Inhibits the activity of gamma -secretase, subunit protein of gamma -secretase or a gamma -secretase-like proteinase (claimed).