Cell-based screening methods
OwnerMichael H. Cardone, Michael Yaffeinventors
Life of this patent
Estimating the term…
Abstract
Cell-based screening methods for determining kinase activity are provided. The methods utilize existing cellular pathways that are regulated by kinases. In one embodiment, various components of a ubiquitin-mediated degradation pathway are modified to create an assay that can be used to screen for a molecule that modulates the activity of a kinase of interest that otherwise does not regulate the degradation pathway. In another embodiment, various components of a protein translocation pathway are modified to screen for a molecule that modulates the activity of a kinase of interest that otherwise does not regulate the translocation pathway.
Claims56
A method for identifying a molecule capable of modulating a kinase activity in situ, said method comprising the steps of:
- exposing a candidate molecule to a cell comprising a phosphorylation substrate having a detectable label associated therewith and a kinase recognition domain altered to be recognized by a kinase that does not recognize said substrate in its unaltered state;
- and
- determining whether said candidate molecule causes a change in an expression of said label, thereby identifying a molecule capable of modulating the activity of said kinase in situ.
A fusion protein comprising an E3 binding region, a kinase recognition domain, and a Green Fluorescent Protein, said kinase recognition domain being selected from the group consisting of kinase recognition domains of β-catenin, HIV protein VPU, p27, Bcl-2, and c-Jun.
A fusion protein comprising an E3 binding region, a kinase recognition domain, and an enzyme capable of producing a detectable enzymatic product, said kinase recognition domain being selected from the group consisting of kinase recognition domains of β-catenin, HIV protein VPU, p27, Bcl-2, and c-Jun.
Dependencies are inferred from the claim text.