Cell-based screening methods
AssigneeMerrimack Pharmaceuticals, Inc.
InventorsMichael H. Cardone, Michael Yaffe
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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.
Claims59
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 enzyme altered to bind a phosphorylation substrate that said enzyme does not bind in its unaltered state, said substrate associated with a detectable label, said binding between the altered signaling enzyme and said substrate being regulated by a kinase;
- and
- determining whether said candidate molecule cause a change in expression of said label, thereby identifying a molecule capable of modulating the activity of said kinase in situ.
A fusion protein comprising a genetically altered signaling enzyme and a label, said alteration producing an adapter module in said signaling enzyme capable of binding to a phosphorylation substrate that said enzyme does not bind in its unaltered state, said binding being regulated by a kinase.
Dependencies are inferred from the claim text.