Fluorescence-based assay for identification of protac ligands for hect-type e3 ligases
AssigneePurdue Research Foundation
InventorsSebastian Kenny, Chittaranjan Das
Life of this patent
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Abstract
A method of identifying a ligand, which is a ligand for a target protein of interest, and which interacts with a homologous to E6AP C-terminus (HECT)-type E3 ligase; a composition comprising (a) a fluorescently labeled peptide comprising a PPxY motif, an E1 ubiquitin (Ub)-activating enzyme, an E2 Ub-conjugating enzyme, a HECT-type E3 ligase, Ub, adenosine triphosphate (ATP), and a reaction buffer or (b) a fluorescently labeled peptide comprising a PPxY motif, a Ub-charged E2 Ub-conjugating enzyme, a HECT-type E3 ligase, ATP, and a reaction buffer; and a composition comprising a ligand identified in accordance with the above method or a pharmaceutically acceptable salt thereof.
Claims7
A method of identifying a ligand, which is a ligand for a target protein of interest and which interacts with a homologous to E6AP C-terminus (HECT)-type E3 ligase, which method comprises:
- (a) incubating the ligand with:
- (i) a fluorescently labeled peptide comprising a PPxY motif and
- (ii) (a′) an E1 ubiquitin (Ub)-activating enzyme, an E2 Ub-conjugating enzyme, the HECT-type E3 ligase, Ub, and adenosine triphosphate (ATP) or
- (b′) a Ub-charged E2 Ub-conjugating enzyme, the HECT-type E3 ligase, and ATP and
- (b) measuring fluorescence polarization (FP) over time, whereupon:
- if the ligand interacts with the HECT-type E3 ligase, the fluorescently labeled PPxY peptide is displaced from binding to the HECT-type E3 ligase, and there is no change in FP over time and
- if the ligand does not interact with the HECT-type E3 ligase, the fluorescently labeled peptide comprising a PPxY motif is ubiquitinated, and there is a change in FP over time,
- whereupon a ligand that interacts with a HECT-type E3 ligase is identified.
A composition comprising (a) a fluorescently labeled peptide comprising a PPxY motif, an E1 ubiquitin (Ub)-activating enzyme, an E2 Ub-conjugating enzyme, a HECT-type E3 ligase, Ub, adenosine triphosphate (ATP), and a reaction buffer or (b) a fluorescently labeled peptide comprising a PPxY motif, a Ub-charged E2 Ub-conjugating enzyme, a HECT-type E3 ligase, ATP, and a reaction buffer.
Dependencies are inferred from the claim text.