In vitro Screening Assay of TCPase Modulators

Abstract

The present invention concerns an in vitro screening assay for identification of modulators of tubulin carboxypeptidase (TCPase) activity comprising the steps of: (i) Contacting (a) a substrate of TCPase enzyme comprising an amino acids sequence having at least the last 4 amino acids residues of the C-terminal sequence of an α-tubulin and/or a Microtubule Associated Protein (MAP) and, as ultimate C-terminal amino acid residue, an aromatic amino acid residue, preferably a tyrosine (Y), and b) an isolated or recombinant TCPase enzyme; in the presence or absence (control) of the modulator compound to be tested, and under conditions for substrate cleavage, preferably detyrosination, and/or liberation of a C-terminal free aromatic amino acid residue, preferably a C-terminal free tyrosine; (ii) Using reagents for detecting and measuring the signal related to substrate cleavage, preferably detyrosination, and/or liberation of the C-terminal free aromatic amino acid residue, preferably the C-terminal free tyrosine; (iii) Measuring and comparing the level of substrate cleavage, preferably detyrosination and/or the level of the C-terminal free aromatic amino acid residue, preferably the C-terminal free tyrosine in presence and in absence (control) of the compound to be tested, and (iv) Selecting the modulators of TCPase for which the level of substrate cleavage, preferably detyrosination or liberation of the C-terminal aromatic amino acid residue, preferably C-terminal free tyrosine is increased in the presence of the compound to be tested (activators of TCPase) or decreased in the presence of the compound to be tested (inhibitors of TCPase). The present invention also concerns kits for performing such in vitro screening assay.

Dependencies are inferred from the claim text.