Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
  • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase

Definitions

• Cellular stress is believed to be an important part of a pathological cellular response. Such cellular stress can be inflicted by several different factors/situations such as oxygen depletion, metabolic dysfunction or chemical compounds. A cellular stress causes the cell to alter its cellular function by an interference with signalling molecules. A prolonged cellular stress can cause disease manifestations in e.g. cardiovascular and metabolic disorders.
• JAK-STAT pathway One of the key signaling pathways in many cells consists of the JAK-STAT pathway.
• Janus Kinases (JAKs) are intra cellular tyrosine kinases and Signal Transducers and Activators of Transcription (STATs) are transcription factors activated by tyrosine phosphorylation and again different molecular forms.
• the JAK-STAT pathway is preferentially activated by the, so-called, cytokine system.
• Cytokines include e.g. growth hormone (GH), prolactin (Prl), leptin, erytropoetin (Epo), colony stimulation factors, different interleukins and interferons.
• GH growth hormone
• Prl prolactin
• Epo erytropoetin
• JAK-STAT pathway activates JAK-STAT pathway through specific membrane bound receptors and it its relevant to note that different forms of JAK molecules exist such as JAK1, JAK2, JAK3 and TYK1. Also different STAT molecules have been isolated, e.g. STAT1, STAT2, STATS, STAT4, STAT5, STAT5a, STAT5b and STAT6. Individual JAK-STAT combinations can be used in a specific signal transduction chain (for reviews, see references 1 and 2).
• the JAK- STAT pathway also includes other signaling molecules e.g. MAP kinases and IRS. According to this invention the JAK-STAT pathway is defined as cellular effects induced by JAK.
• SOCS Cytokine Signaling
• the present invention relates to an assay system, based on the JAK-STAT-SOCS pathway, which can be used to monitor compounds that cause a cellular stress.
• Cellular stress can be monitored in several other ways e.g. by measuring heat shock response proteins or apoptosis.
• the analysis of the JAK-STAT-pathway in relation to actions of chemical compounds is advantageous as the JAK-STAT-pathway is an important intra cellular pathway and pharmacological compounds can influence the JAK-STAT pathway without causing overt heat shock responses and apoptosis.
• the technology described in the present invention can be used to screen chemical, biological, and physical entities, and particularily established drugs or potential drug candidates, for their effects on the JAK-STAT-SOCS pathway. If a particular compound scores positive in this assay it may cause cellular stress, which might lead to side effects if intact organisms are treated with said compound.
• JAK-STAT-SOCS pathway As exemplified below an analysis of the JAK-STAT-SOCS pathway can be used to reveal potential side effects of test compounds.
• the bearing principle is to first activate a JAK- STAT-SOCS pathway in a cell and then add a test compound.
• many factors can activate the JAK-STAT pathway in cultured cell and the actual nature of these, as well as doses required, are known to any person skilled in the art.
• factors that activate the JAK-STAT pathway include growth hormone, prolactin, erytropoetin, leptin, erythropoetin, a variety of interleukins, colony stimulation factors and interferons.
• JAK-STAT pathway there are several different methods available to detect components of the JAK-STAT pathway, these include protein analysis of activated, i.e. phospho- rylated, JAKs or STATs as well as the measurement of levels of SOCSs with antibody techniques or related protein-binding techniques.
• Alternative assays are gel shift analysis or similar DNA binding techniques that can be used to detect STAT DNA binding and different types of hybridisation techniques that can be used to analyse SOCS mRNA levels.
• genetic tagging techniques where components in the JAK-STAT-SOCS pathway are modified with a tag to simplify detection in the cell are applicable. The present invention can thus be carried out using a variety of methods.
• JAK-STAT pathway or JAK-STAT system is herein defined as any cellular event that depends on the activation of JAK.
• JAKs or STATs that can be used to measure a cytokine signal such as activation of MAP kinases and IRS.
• the principle of the invention relies in the use of isolated cells in which the JAK-STAT pathway is activated. In case a cell is exposed to a test compound the interference of such a test compound with the JAK-STAT pathway will be detected by a temporal analysis of JAK phosphorylation or by STAT-5 DNA binding (or alternative methods) or by a temporal analysis of SOCS protein/mRNA levels.
• the term "temporal analysis” means a comparison of the duration of the JAK-STAT signal at the activation of the JAK-STAT pathway in the presence of a biologically active entity with the duration of a JAK-STAT signal caused by a cytokine in the absence of a biologically active entity. A significant change, normally a prolongation of the duration of the JAK-STAT pathway is assayed for.
• the use of the different end point read outs, here exemplified as STAT activation and SOCS levels can be converted into high through-put assays by a person skilled in the art.
• cells can be grown, preferably in a 96 well format, then exposed to a cytokine such as growth hormone and either the STAT activation or levels of SOCS measured.
• a cytokine such as growth hormone
• STAT activation or levels of SOCS measured.
• Initial experiments are needed to establish the time kinetics for cytokine activation of the JAK-STAT pathway with regard to the particular cytokine used and the particular cell line used. It is to be expected that the activation is transient which means that one time point where the JAK-STAT pathway is maximally active can be identified and another, later, time point, where the signal has been significantly reduced.
• GH is used as the stimulatory cytokine in liver cells the activation of the JAK-STAT pathway is maximal within a time period of one hour (lh) and the signal is significantly reduced after four hours (4h).
• test compounds can be added in conjunction with the activation of the JAK-STAT pathway.
• a suitable time period to analyse components of the JAK-STAT pathway in the GH system can be 4h. At this time period one can assay for a prolongation of the JAK-STAT pathway consisting of e.g. maintained STAT DNA binding and/or JAK phosphorylation and/or levels of SOCS mRNA or SOCS protein.
• cell types i.e. derived from different cell linages
• cells can be of different embryo- logical origin such as from endoderm, ektoderm, mesenchyme and so forth.
• Primary cell cultures as well as established cell lines can be used. The main features will be to use cells commonly used in the laboratory and a list of such cells can be obtained from the ATCC (American Tissue Culture Collection). In certain cases it might be useful to analyse cells, e.g. fibro- blasts or tumor cells, from individual patients to characterise the response to chemical compounds.
• the model has been JAK2 activation of STAT-5 in liver cells; a prolongation of STAT-5 DNA binding has been the read out for test compounds or a reduction of SOCS 2, SOCS 3 and CIS.
• the time kinetics as well as the model used in the examples are only meant as illustrations of the invention and are not intended to limit the use of this invention. Variations in experimental designs such as the used cell, the time kinetics applicable and/or the component of the JAK/STAT/SOCS pathway used as an end point measurement, fall within the scope of the present invention as long as the screening system is cell based, a cell in which the JAK-STAT-SOCS system is activated used and a temporal analysis of the effects of the test compounds on this pathway conducted.
• Figure 1 relates to the transient nature of STAT5 activation by GH measured using Gel mobility shift assay.
• BRL4 cells were grown for 8 hours under serum free conditions. Recombinant hGH (50nM) was then added, the cells were harvested at the indicated points of time and nuclear extracts were prepared. STAT5 and STAT1 binding activity were determined using electrophoresis mobility shift assay with a 32 P-labeled SPIGLE1 probe. Relative DNA binding levels were measured by autoradiography. This experiment shows that GH activates STAT5 DNA binding in a transient fashion. After GH stimulation a STAT DNA binding activity is rapidly increased followed by a rapid decrease to background level after 2 hours.
• BRL-4 cells (a rat liver cell) that respond to GH were used (5).
• GH was added to these cells at a concentration of 100 mg/ml. As shown in Fig 1 this cause a transient activation of STAT-5 measured in a gel shift analysis (6); a maximum STAT-5 binding was observed after 30 minutes and after 4h the response was lost.
• a large variety of chemical compounds were tested for their ability to prolong STAT-5 activation and a surprising finding was that so may compounds could prolong a STAT-5 activation in the presence of GH. In the absence of GH these compounds did not exert any effect. As listed in Table 1 such compounds included CHX, D609, Bapta and H7.
• One embodiment of the present invention is a cell based drug screen by which a drug might be categorized for a possible interaction with the JAK-STAT-SOCS pathway.
• a cytokine receptor would here be activated in a cell by one cytokine or a combination of different cytokines.
• Such cytokines could consist of proteins from varying sources and of varying purity, protein variants of cytokines or possibly of analogues of cytokines.
• the drug of interest is added in conjunction with (before, together or after) the cytokine stimulation. Subsequently the effect of said drug could be detected by analysing the STAT proteins in a period of time. Interference in the pathway will result in a prolongation of a cytokine response on STAT DNA binding activity or a prolongation of the presence of STAT tyrosine phosphorylation.
• assays can be conducted in an automated or semi-automated fashion suitable for screening purposes. 2.
• the SOCS protein levels is subsequently measured. This can be achieved with different types of e.g. protein binding assays. In a screening assay, test compounds will score positive if they cause a reduction of SOCS proteins including SOCS1, SOCS2, SOCS3 and CIS.

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• 1999
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  • 2000-10-27 EP EP00976484A patent/EP1226268A1/en not_active Withdrawn
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