Cell-based screening methods
OwnerMichael H. Cardone, Michael Yaffeinventors
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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.
Claims33
A fusion protein comprising a pair of sequestering motifs, an enzyme-binding region, and a label, said pair of sequestering motifs flanking each side of said enzyme-binding region, said enzyme-binding region being prevented from binding a signaling enzyme when said sequestering motifs interact with each other, said interaction between the sequestering motifs being regulated by a kinase.
A cell transfected with said vector of claim 17.
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 signaling substrate comprising an enzyme-binding region flanked on both sides by a pair of sequestering motifs, said enzyme-binding region being prevented from binding a signaling enzyme when said sequestering motifs interact with each other, said interaction between the sequestered motifs being regulated by a kinase, said signaling substrate further associated with a detectable label;
- and
determining whether said candidate molecule causes a change in expression of said label, thereby identifying a molecule capable of modulating the activity of said kinase in situ.
Dependencies are inferred from the claim text.