Substrates and assays for β-secretase

A method for assaying for modulators of β-secretase activity, comprising:

• (a) contacting a polypeptide with β-secretase APP processing activity with a substrate, both in the presence and in the absence of a putative modulator compound;
• wherein said substrate comprises a peptide having an amino acid sequence of at least 6 amino acids, said amino acid sequence including four amino acids defined by formula P2P1-P1′P2′, wherein:
• P2 is N;
• P1 comprises an amino acid selected from the group consisting of Y, L, and F;
• P1′, comprises an amino acid selected from the group consisting of E, A, and D;
• P2′, is V;
• wherein the substrate is cleaved between P1 and P1′ by a human aspartyl protease encoded by the nucleic acid sequence of SEQ ID NO:
• 1 or SEQ ID NO:
• 3 (Hu-Asp2);
• and
• wherein said peptide does not comprise the corresponding P2P1-P1′P2′ portion of amino acid sequence depicted in SEQ ID NO:
• 34;
• (b) measuring cleavage of the substrate peptide in the presence and in the absence of the putative modulator compound;
• and
• (c) identifying modulators of β-secretase activity from a difference in substrate cleavage in the presence versus in the absence of the putative modulator compound, wherein a modulator that is a β-secretase antagonist reduces such cleavage and a modulator that is a β-secretase agonist increases such cleavage.