Abstract: A method is described to assay for protein interactions in living cells, e.g. by the introduction of two heterologous conjugates into the cell. The method uses the measurement of cellular distribution of a detectable component (e.g. a GFP-labeled˜fluorescent probe) to indicate the presence or absence of an interaction between that component and a second component of interest. The method uses the knowledge that certain components can be stimulated to redistribute within the cell to defined locations. Inducible redistribution systems make it possible to determine if specific interactions occur between components. Inducible systems are described where it is demonstrated that the redistribution stimuli are essentially “null”, in that they affect no other system in the cell during the assay period, other than the component whose redistribution can be induced.

Abstract: A method and apparatus for screening an array of test compounds for bioactivity by contacting an array of test compounds with a detector layer capable of detecting bioactivity, and detecting a detector layer response. The detector layer is comprised of physiologically viable cells. The method and apparatus allow a large number of test compounds to be simultaneously assayed in parallel without the need for complex fluidic devices.

Abstract: A method and apparatus for screening an array of test compounds for bioactivity by contacting an array of test compounds with a detector layer capable of detecting bioactivity, and detecting a detector layer response. The detector layer is comprised of physiologically viable cells. The method and apparatus allow a large number of test compounds to be simultaneously assayed in parallel without the need for complex fluidic devices.

Abstract: A method is described to assay for protein interactions in living cells, e.g. by the introduction of two heterologous conjugates into the cell. The method uses the measurement of cellular distribution of a detectable component (e.g. a GFP-labelled fluorescent probe) to indicate the presence or absence of an interaction between that component and a second component of interest. The method uses the knowledge that certain components can be stimulated to redistribute within the cell to defined locations. Inducible redistribution systems make it possible to determine if specific interactions occur between components. Inducible systems are described where it is demonstrated that the redistribution stimuli are essentially “null”, in that they affect no other system in the cell during the assay period, other than the component whose redistribution can be induced.

Abstract: Fluorescence complementation products with intensity levels mimicking the full length intensities are obtained by introduction of improved folding capabilities with a mutation in position 1 preceding the chromophore. This is particularly seen with the yellow variant of Green Fluorescent Protein (GFP). An additive increase is obtained by splitting the GFP between amino acids 172 and 173. Screening for drugs capable of preventing interaction between proteins is performed by selecting the cells with the highest dynamic range through Fluorescence Activated Cell Sorting (FACS), as illustrated with the ability of FK506 to break the rapamycin induced interaction between FRB and FKBP.

Abstract: An alternative therapeutic approach for PDE4 inhibition is disclosed. PDE4 dislocators, will remove the PDE4 away from the native location in the cell and thereby increase the concentration of cAMP in this location. By dislocating the PDE4, and thereby not acting directly on the catalytic, among phosphodiesterase inhibitors, well conserved site, the compound will act e.g. at the binding domain of the PDE4, thereby providing isoform-specific ‘inhibitors’ of PDE4. The dislocation of PDE4s are visualised with fusions to GFP. The native location is induced by treatment with Rolipram.

Abstract: The present invention relates to a 3-part hybrid system for detection protein interactions in live mammalian cells and screening for compounds modulating such interactions. The method is fully compatible with HTS. The three hybrids are a first heterologous conjugate comprising an anchor protein that specifically binds to an internal structure within the cell conjugated to an interactor protein of type A, a second heterologous conjugate comprising an interactor protein of type B conjugated to the first protein of interest, a third heterologous conjugate comprising a second protein of interest conjugated to a detectable group. When applying a dimerizer compound, interactor proteins A and B bind to each other and if the two proteins of interest interact, the distribution of the detectable group will mimic the distribution of the anchor protein. However, if there is no interaction, the distribution of the detectable group will mimic the distribution of the second protein of interest.