Chemical genetic strategies to delineate MAP kinase signaling pathways using protein-fragment complementation assays (PCA).

Signal transduction pathways mediated by MAP kinases are among the most studied. Direct analysis of MAP kinase pathways has been difficult because some details of MAP kinase signaling cannot be studied in vitro. Here, we describe a strategy for directly analyzing MAP kinase signaling pathways in living cells using protein-fragment complementation assays (PCA) based on intensely fluorescent proteins.

Exploiting network biology to improve drug discovery.

Mammalian signal transduction occurs in the context of multiprotein complexes, yet currently available drug discovery strategies do not reflect this fact. We present a strategy for screening drugs and targets in living human cells by utilizing high content protein-fragment complementation assays. Synthetic fragments of a mutant fluorescent protein ("Venus" and/or enhanced yellow fluorescent protein) are used for protein-fragment complementation assay construction, allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways.

Identifying off-target effects and hidden phenotypes of drugs in human cells.

We present a strategy for identifying off-target effects and hidden phenotypes of drugs by directly probing biochemical pathways that underlie therapeutic or toxic mechanisms in intact, living cells. High-content protein-fragment complementation assays (PCAs) were constructed with synthetic fragments of a mutant fluorescent protein ('Venus', EYFP or both), allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. One hundred and seven different drugs from six therapeutic areas were screened against 49 different PCA reporters for ten cellular processes.