Novo Nordisk A/s

Fisher Bioimage Aps

Bioimage A/s

2curex

2curex Aps

Norrild, Jens

Thastrup, Ole

Ole Thastrup

Lars Kongsbak Poulsen

Kurt Scudder

Grith Hagel

Sara Petersen Bj&oslash;rn

Sara Petersen Bjørn

Lars Christian Petersen

Lars Kongsbak

Bernard Robert Terry

Sara Petersen Bjorn

Don Foster

Niels Bergenhem

S&oslash;ren Tullin

Len Pagliaro

Kasper Almholt

Soren Tullin

Jens Chr Norrild

Morten P Meldal

Agneta Siegbahn

Mirella Ezban

Søren Tullin

Soren Jensby Nielsen

Per Olaf Gunnar Arkhammer

Morten Hentzer

Jaromir Ruzicka

Sara Petersen Bjo Slashedrn

So Slashedren Tullin

Jens Norrild

The present invention relates to a method and tools for extracting information on a compounds influence on a cellular phenotype. The method of the invention may be used as a very efficient procedure for testing the efficacy or resistance of single drugs or combinations of drugs on cells from individual patients. Thus, the methods may be useful for predicting efficacy of a drug on a given patient. The methods are also useful for testing of compounds for toxicity, identifying drug targets for known or novel compounds.

Identifying compounds modifying a cellular phenotype

The present invention relates to methods for identifying compounds capable of modulating a cellular response. The methods involve attaching living cells to solid supports comprising a library of test compounds. The test compounds are linked to the solid support via cleavable linkers and may thus be released from the solid supports. Solid supports comprising cells, wherein the cellular response of interest has been modulated are selected and the test compound of the solid support can then be identified. The cellular response may for example be changes in complex formation between proteins.

Identification of compounds modifying a cellular response

The present invention relates to compounds capable of inhibiting binding of the Smac protein to Inhibitors of apoptosis (IAPs). Such compounds are preferably capable of inhibiting IAP and thus may promote apoptosis or sensitize cells for apoptosis. The compounds may be used in the treatment of proliferative diseases, such as cancer.

Compounds modifying apoptosis

Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tyrosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signalling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of the influence. Measurement of redistribution is performed by recording of light intensity, fluorescence lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a fluorescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating a disease related to the function of the component.

Method for extracting quantitative information relating to an influence on a cellular response

A GFP with an F64L mutation and E222G mutation is provided. This GFP has a bigger Stokes shift compared to other GFPs making it very suitable for high throughput screening due to a better resolution. This GFP also has an excitation maximum between the yellow GFP and the cyan GFP allowing for clearer band separation when used together with those GFPs. Examples include the sequences in SEQ ID NOs: 3 and 4.

Nucleic acids encoding fluorescent proteins and methods of using the same

The present invention relates to novel variants of the fluorescent protein GFP having improved fluorescence properties.

Fluorescent proteins

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&#x2dc;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. Also described is an extraction buffer which is useful in high throughput screening for drugs which affect the intracellular distribution of intracellular components. The extraction buffer comprises a cellular fixation agent and cellular permeabilisation agent. Optimizing the composition of the extraction buffer and its application to various cell types is described.

Method for extracting quantitative information relating to interactions between cellular components

A GFP with an F64L mutation and an E222G mutation is provided. This GFP has a bigger Stokes shift compared to other GFPs making it very suitable for high throughput screening due to a better resolution. This GFP also has an excitation maximum between the yellow GFP and the cyan GFP allowing for cleaner band separation when used together with those GFPs.

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.

Method and apparatus for high density format screening for bioactive molecules

The present invention relates to a method of detecting biologically active substances affecting intracellular processes, an isolated DNA, a vector and a cell for use in the method. More specifically, the present invention provides a method for detecting a biologically active substance that affects intracellular processes mediated through a protein kinase or a second messenger, which comprises incubating a cell with a test substance and measuring any change caused by the test substance in the flourescence of (i) a wild-type or modified green flourescent protein (GFP) having a protein kinase recognition site, (ii) a modified GFP containing a second messenger binding domain, or (iii) a hybrid polypeptide having a wild-type or modified GFP and an attached protein kinase recognition site or a second messenger binding domain.

Method of detecting biologically active substances

Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Flourescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP- or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine/threonin protein kinase, a tryosine protein kinase, or a protein phosphatase (e.g. PKA, PKC, Erk, Smad, VASP, actin, p38, Jnkl, PKG, IkappaB, CDK2, Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signaling pathway in such a way that the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of influence. Measurement of redistribution is performed by recording of light intensity, flourescence lifetime, polarization, wavelength shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a flourescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating a disease related to the function of the component.

The present invention provides methods for modifying the motility of tissue factor (TF)-expressing cells, which are carried out by contacting such cells with an effective amount of Factor VIIai Factor VIIa agonist.

Methods for modifying cell motility using factor VIIa or inactivated factor VIIa

The present invention provides methods for identifying candidate drugs that modulate factor VIIa-mediated intracellular signaling by measuring the effects of such drugs on the activation of the mitogen-activated protein kinase (MAP kinase) signaling pathway.

Identification of drug candidates modulating factor VII-mediated intracellular signaling

The present invention provides a method for detecting a biologically active substance that affects intracellular processes mediated through a protein kinase or second messenger, which comprises incubating a cell with a test substance and measuring any change caused by the test substance in the fluorescence of (i) green fluorescent protein (GFP) having a protein kinase recognition site or (ii) GFP that has been modified to contain a second messenger binding domain.

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Birkerød, Denmark

Ole Thastrup