WO2001088194A1 - Procedes et compositions destines a l'amplification de la transduction de signaux intracellulaires - Google Patents

Procedes et compositions destines a l'amplification de la transduction de signaux intracellulaires Download PDF

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WO2001088194A1
WO2001088194A1 PCT/US2001/015426 US0115426W WO0188194A1 WO 2001088194 A1 WO2001088194 A1 WO 2001088194A1 US 0115426 W US0115426 W US 0115426W WO 0188194 A1 WO0188194 A1 WO 0188194A1
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cell
nucleic acid
reporter
control element
protein
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PCT/US2001/015426
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Erik A. Whitehorn
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Whitehorn Erik A
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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention relates to the field of drug discovery and cellular signal transduction.
  • cells including prokaryotic and eukaryotic cells, have evolved mechanisms for detecting an extracellular signal and transducing that signal to form an intracellular signal which appropriately influences various intracellular activities.
  • signals can affect the growth rate of cells, activate DNA repair mechanisms and regulate transcription of genes.
  • signal transduction is effectuated via cellular receptors that bind chemical stimuli specifically and generate an internal signal.
  • Many receptors are located at the cell surface and include an extracellular domain to interact with a stimulus, a transme brane domain and an intracellular domain.
  • Other receptors are intracellular proteins, such as nuclear receptors and receptors for steroid hormones.
  • Signal transduction typically involves a ligand mediated structural change in the receptor that triggers a cascade of cellular events that modify/activate a number of proteins, some of which direct the expression of certain genes.
  • the nicotinic type acetylcholine receptor is a membrane bound ion channel that opens a pore within the protein to allow an influx of sodium ions into the cell in response to binding of acetylcholine.
  • Many receptors transduce a signal via different secondary messengers that are part of a cellular cascade.
  • One ofthe more common secondary messengers is cyclic AMP (cAMP).
  • cAMP cyclic AMP
  • certain receptors upon binding of a ligand, activate the intracellular enzyme adenylate cyclase. This enzyme catalyzes the synthesis of cAMP, which in turn activates other components ofthe signal transduction process.
  • cAMP activation has been established. Once formed within the cell, cAMP acts via regulatory elements associated with cAMP-inducible genes. Certain aspects of cAMP activation have been established. Once formed within the cell, cAMP acts via regulatory elements associated with cAMP-inducible genes. These response elements are typically referred to as CREs (cAMP-response elements), and are binding sites for transcription factors.
  • CREs cAMP-response elements
  • One step in cAMP activation involves phosphorylation of a protein kinase to form an activated form. The activated protein kinase then phosphorylates a transcription factpr called CREB (c AMP-response element binding protein) to convert it into active form.
  • CREB activation does not affect its DNA binding capabilities; rather, the transcriptional activation function of the protein is activated.
  • the JAK /STAT (an acronym for "Janus tyrosine kinase/signal transducers and activators of transcription") signal transduction pathway is another signal cascade that is common to various receptors, such as cytokines for example.
  • Receptors involved in this pathway typically are single transmembrane proteins.
  • various tyrosine kinase reactions result in the activation of proteins associated with the receptor on the cytoplasmic side (the JAK proteins).
  • the activated JAK proteins in turn phosphorylate a STAT protein to convert it into active form.
  • This active form can then bind to a SRE (STAT response element) nucleotide binding segment to activate transcription (see, e.g., Darnell Jr., J.E., Science, 277:1630-1635 (1997); and Lamb, P., et al., Drug Discovery Today 3:122-130, (1998), both of which are incorporated by reference in their entirety).
  • SRE STAT response element
  • Another general signal transduction pathway grouping is called the MAP (Mitogen Activated Protein)-Kinase pathway.
  • MAP Mitogen Activated Protein
  • This pathway broadly refers to pathways that respond to a variety of signal inputs, but generally include serine and threonine kinases. The pathways are typically multistep pathways and include many branchpoints.
  • MAP Kinase response elements are also referred to as SREs. In this instance, however, the acronym stands for service response elements. Such pathways are described, for example, by Schaeffer, H. J. and Weber, M. J.,
  • the present invention provides methods and compositions for identifying or assaying compounds that influence signal transduction within cells.
  • the methods and compositions are useful in assaying for compounds that interact with a component of a signal cascade.
  • the methods and compositions can be used to identify or assay agonists or antagonists that bind tp a receptor or compounds that activate or inhibit other components of a signal cascade.
  • the methods of the present invention involves two processes. Initially, an intracellular signal triggers the formation of a transactivator protein. The transactivator protein then binds to a cognate control element to activate transcription of reporter that can be detected. This two-step approach can achieve a significant increase in signal strength relative to systems that do not utilize a transactivator to activate transcription of reporter.
  • Certain methods provided by the invention are general methods for assaying compounds that activate signal transduction. Some of these methods involve contacting a cell having a receptor with a compound to generate an intracellular signal.
  • the cell that is contacted comprises: (i) a first heterologous nucleic acid comprising a first transcriptional control element operatively linked to a nucleic acid segment encoding a transactivator protein that comprises a DNA binding domain and a transcriptional activation domain; and (ii) a second heterologous nucleic acid sequence comprising a second transcriptional control element and a segment that encodes a reporter.
  • the second transcriptional control element can interact with the DNA binding domain of the transactivator protein and is operatively linked to the segment encoding the reporter.
  • the transactivator protein then interacts with the second transcriptional control element to activate expression of the reporter. Expression of the reporter is subsequently detected. Detection of reporter expression can involve, for example, detection of the formation of transcript or translation product.
  • screening methods are designed to screen for a compound that affects a component of a signal cascade.
  • Some screening methods involve contacting the test cell that can trigger the signal cascade with a test compound.
  • the test cell comprises: (i) a first heterologous nucleic acid comprising a first transcriptional control element operatively linked to a nucleic acid segment encoding a transactivator protein that comprises a DNA binding domain and a transcriptional activation domain; and (ii) a second heterologous nucleic acid comprising a second transcriptional control element that can interact with the DNA binding domain of the transactivator protein and operatively linked to a nucleic acid encoding a reporter.
  • a component of the signal cascade activates transcription via the first transcriptional control element to express the transactivator protein.
  • the transactivator protein then interacts with the second transcriptional control element to activate expression of the reporter to produce a basal level of reporter expression.
  • the test compound interacts with a component of the signal cascade, it modulates the basal level of reporter expression.
  • a modulation in reporter expression is detected, a change indicating that the test compound interacts with a component of the signal cascade.
  • the level of expression of reporter can be compared to reporter expression in a control cell that is identical to the test cell but that is not contacted with the test compound. A difference in expression of the reporter between the test and control cell indicating that the test compound affects a component of the signal cascade.
  • test cell comprises: (i) a receptor that can transduce an extracellular signal to form an intracellular signal within the test cell, (ii) a first heterologous nucleic acid comprising a first transcriptional control element operatively linked to a nucleic acid segment encoding a transactivator protein that comprises a DNA binding domain and a transcriptional activation domain; and (iii) a second heterologous nucleic acid sequence comprising a second transcriptional control element that can interact with the DNA binding domain of the transactivator protein and operatively linked to a nucleic acid encoding a reporter.
  • the intracellular signal activates transcription via the first transcriptional control element to express the transactivator protein.
  • the transactivator protein then interacts with the second transcriptional control element to activate expression of the reporter. Expression of the reporter is detected, expression of reporter indicating that the test compound affects signal transduction.
  • the first cell also comprises (i) a first heterologous nucleic acid comprising a first transcriptional control element operatively linked to a nucleic acid segment encoding a transactivator protein that comprises a DNA binding domain and a transcriptional activation domain; and (ii) a second heterologous nucleic acid sequence comprising a second transcriptional control element that can interact with the DNA binding domain of the transactivator protein and operatively linked to a reporter sequence.
  • the intracellular signal generated by the known compound activates transcription via the first transcriptional control element to express the transactivator protein.
  • the transactivatpr protein then interacts with the second transcriptional control element to activate expression of the reporter.
  • a second cell identical to the first cell is contacted with the known compound under the same conditions as the second cell, except that the second cell is also contacted with a test compound that is a potential antagonist. Expression of the reporter from the first and second cell is detected, a reduction in the level of expression in the second cell relative to the first cell indicating that the test compound is a potential antagonist of the receptor.
  • the invention also provides nucleic acids that comprise a transcriptional control element operatively linked to a sequence encoding a fusion protein that comprises a prokaryotic DNA binding domain and an eukaryotic transcriptional activation domain, wherein the transcriptional control element is responsive to a transduced signal within a cell.
  • Vectors and cells including such a nucleic acid are also provided.
  • Certain cells include a receptor that can transduce an extracellular signal to form an intracellular signal within the cell and two heterologous nucleic acids.
  • One heterologous nucleic acid comprises a first transcriptional control element operatively linked to a nucleic acid segment encoding a transactivator protein that comprises a DNA binding domain and a transcriptional activation domain, wherein the first transcriptional control element can activate the transcription of the segment encoding the transactivator in response to the intracellular signal.
  • the second nucleic acid comprises a second transcriptional control element operatively linked to a nucleic acid segment encoding a reporter, wherein the transactivator protein interacts with the second transcriptional control element via its DNA binding domain and directly or indirectly activating transcription of the reporter segment via its transcriptional activation domain.
  • kits including the two nucleic acids just described for introduction into cells.
  • the kits can also include cells into which the nucleic acids can be transfected.
  • CPS abbreviation CPS used in the following figures stands for counts per second and is a measure of luminescence intensity resulting from reporter (luciferase) activity.
  • FIG. 1 is a schematic of elements and events for a specific example of a two- step JAK/STAT signal transduction system.
  • FIGS. 2A and 2B depict specific examples of plasmid constructs for JAK/STAT based signal transduction systems (pEWP-LuxRE:8X9G for one-step systems, and pTRLuxRE:8X9G for two-step systems).
  • the plasmids respond to STAT activation through the 8X9G binding domain.
  • the two step plasmid includes a Tta transactivator and a 7x Tet operator as the transcriptional control element to control reporter expression.
  • FIGS. 3 A and 3B depict specific examples of plasmid constructs for MAP- K Elk-1 based signal transduction systems (pGa!4-Elk-l for one-step systems, and pTRLux:Gal4-Elk-l for two-step systems).
  • the plasmids respond to Gal4 activation through the Gal4 DNA binding domain.
  • the two-step plasmid (pTRLux:Gal4-Elk-l) includes a Tta transactivator controlled through the Gal4 DNA binding domain and a 7x Tet operator as the transcriptional control element to control reporter expression.
  • FIGS. 4A and 4B depict specific examples of plasmid constructs for use in CREB based signal transduction systems (pEWP 6 CRE-TK Lux for one-step systems and pTR Lux for two-step systems).
  • the plasmids respond to CREB activation through a CRE transcriptional control element (6 CRE).
  • the two-step plasmid pTR Lux includes a Tta transactivator controlled through the 6 CRE DNA binding domain and a 7x Tet operator as the transcriptional control element to control reporter expression.
  • FIG. 4C depicts one specific example of a plasmid construct for use in a
  • FIGS. 5A-5D show results for a JAK/STAT based signal transduction system in HepG2 cells harboring the single- (pEWP-Lux RE-8X96) and two-step (pTRLuxRE:8X9g) reporter constructs (see FIGS.
  • FIGS. 5A-5B present dose response curves for a JAK/STAT based signal transduction system with single- and two-step reporter constructs, respectively. Comparable EC50s for both ligands OSM and IFN ⁇ result for both types of reporter constructs.
  • FIG. 5C depicts signal maxima and fold increases for the one-step and two-step reporter systems when cells were incubated with OSM or gamma interferon IFN ⁇ (100 ng/ml).
  • FIG. 5D depicts tetracycline regulation of HepG2 cells harboring the two-step construct pTRLuxRE:8X9g (see FIG. 2B).
  • Cells were stimulated with 100 ng/ml oncostatin M (OSM) for 6 hours after a 16 hour incubation with varying amounts of tetracycline. Tetracycline concentrations were maintained during the assay.
  • OSM oncostatin M
  • FIGS. 6A-6C show results of experiments conducted with a JAK/STAT based signal transduction system similar to that used to conduct experiments shown in FIGS. 5A- 5D, except with BaF3 cells expressing an endogenous JJ -3 cytokine receptor instead of HepG2 cells (constructs shown in FIGS. 2A and 2B). Reporter expression in the two-step system required binding of a tTA transactivator to a tet operator transcriptional control element.
  • FIGS. 6 A and 6B show induced stimulation of the BaF3 cells with the cytokine JJ - 3 (10 ng/ml).
  • FIG. 6A the Y-axis is a linear scale
  • FIG. 6B the scale is loglO to more clearly show the one-step response
  • FIG. 6C is a graph showing the effect of tetracycline (l ⁇ g/ml) on ligand induced reporter activation of BaF3 cells expressing the human thrombopoietin (TPO) receptor and harboring the, two-step construct.
  • FIGS. 7A-7B depict results of experiments with a MAP-K transduction pathway system. Experiments were conducted with CHO cells expressing the human insulin receptor and harboring the plasmid pGal4-Elk-l (one-step system) or the plasmid ⁇ TRLux:Gal4-Elk- 1 (two-step system) (see FIGS. 3A and 3B). The two-step system included a tTA transactivator and a tet operator as a transcriptional control element.
  • FIG. 1 The two-step system included a tTA transactivator and a tet operator as a transcriptional control element.
  • FIG. 7A is a dose response curve for the two types of cells at varying insulin concentrations.
  • FIG. 7B is a plot showing a comparison of signal maxima and fold increases in signal for the two types of cells at 0 or 1 ⁇ g/ml insulin.
  • FIGS. 8A-8D show results of experiments conducted with a CRE/CREB mediated signal transduction system.
  • FIGS. 8 A and 8B show inhibition of signal generation by tetracycline in CHO cells expressing the CCKb receptor with one-step and two-step constructs (see FIGS. 4 A and 4B, respectively). Signal was generated by incubation of the cells with 100 ng/ml CCK8 after overnight incubation in varying concentrations of tetracycline.
  • FIGS. 8C and 8D depict dose response curves for one- and two-step systems, respectively.
  • a CCK8 dose response curve of cells with these two systems show equivalent EC 50s (6.8 nM for one-step and 7.9 nM for two-step) but differences in the fold increases of generated signals (10X for the one-step and 7X for the two-step) and comparative signal amplitude (two-step creates a 38 fold greater signal than the one-step).
  • nucleic acid or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acids are chemical analogues of a corresponding naturally- occurring amino acids:- -
  • operably linked generally refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid or nucleic acid segment is operatively linked when it is placed in functional relationship to another nucleic acid or nucleic acid segment.
  • the term refers, for example, to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, transcriptional control element or array of transcription factor binding sites) and a second polynucleotide, wherein the expression control sequence affects transcription and/or translation of the second polynucleotide.
  • a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • heterologous sequence or a “heterologous nucleic acid,” as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
  • a heterologous gene in a prokaryotic host cell includes a gene that, although being endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous nucleic acid.
  • Recombinant when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid.
  • Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell.
  • Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means.
  • the term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such ⁇ modifications include those obtained by gene replacement, site-specific mutation, and related techniques.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature and which has not been intentionally modified by humans in the laboratory is naturally-occurring.
  • a "tetracycline analog” includes compounds related to tetracycline that can bind to the tetracycline repressor with a K a of at least 10 6 M “1 , preferably 10 9 M “1 or greater.
  • Examples of such analogs include, but are not limited to, those described by HJavka and Boothe, "The Tetracyclines," in Handbook of Experimental Pharmacology 78, R.K. Blackwood et al (eds.), Springer- Veriag, Berlin-New York, 1985; by Mitshef, L.A., "The
  • a “carbohydrate” refers to aldehyde or ketone derivatives of polyhydric alcohols.
  • the term includes monosaccharides, oligosaccharides and polysaccharides.
  • Oligosaccharides contain a relatively limited number of monosaccharide residues, and typically include di-, tri-, tetra- and pentasaccharides.
  • Polysaccharides are polymers of high molecular weight formed from the condensation of many monosaccharides of the same type (homopolysaccharides) or two or more types (heteropolysaccharides).
  • the molecular weight of polysaccharides can range intp the millions of daltons.
  • Specific examples of carbohydrates include glucose, galactose, xylose, fructose, sucrose, and glycogen.
  • sugar typically refers to monosaccharides.
  • lipid generally refers to substances that are extractable from animal or plant cells by nonpolar solvents. Materials falling within this category include the fatty acids, fats such as the mono-, di- and triacyl glycerides, phosphoglycerides, sphingolipids, waxes, terpenes and steroids. Lipids can also be combined with other classes of molecules to yield lipoproteins, lipoamino acids, lipopolysaccharides and proteolipids.
  • “Fatty acids” generally refer to long chain hydrocarbons (e.g., 6 to 28 carbon atoms) terminated at one end by a carboxylic acid group, although the hydrocarbon chain can be as short as a few carbons long (e.g., acetic acid, propionic acid, n-butyric acid). Most typically, the hydrocarbon chain is acyclic, unbranched and contains an even number of carbon atoms, although some naturally occurring fatty acids have an odd number of carbon atoms. Specific examples of fatty acids include caprioic, lauric, myristic, palmitic, stearic and arachidic acids. The hydrocarbon chain can be either saturated or unsaturated.
  • Fats are a particular class of lipids and are esters of fatty acids and glycerol. Fats include mono-, di ⁇ - and tri-acylglycerides.
  • a “nucleoside” is a compound of a sugar (typically a ribose or deoxyribose) attached to a purine or pyrimidine base via an N-glycosyl linkage.
  • a “nucleotide” refers to a phosphate ester of pentose sugars in which a nitrogenous base (typically a purine or pyrimidine base) is linked to the C(l') sugar residue. Most typically, a nucleotide is a nucleoside attached to a phosphoric group.
  • steroid refers to the large class of compounds that contain the tetracyclic cyclopenta[ ⁇ ]phenanthrene backbone that are part ofthe metabolism of an organism.
  • a specific example is cholesterol.
  • the invention provides methods, cells, nucleic acids and kits for identifying or assaying compounds that affect cellular signal cascades by interfering or potentiating steps within a cascade, for example. Certain methods, compositions and kits are useful in identifying or assaying agonists or antagonists of receptors, which compounds are capable of generating or affecting intracellular signals that regulate transcription. The methods and compositions of the invention amplify such intracellular signals through a cascade process to produce an amplified signal that can be readily detected.
  • the invention is broadly applicable to assaying for test compounds that interact with one or more components of the cascade, thereby interfering or activating the cascade.
  • the methods of the invention generally involve contacting a cell with a test compound. If the test compound interacts with a component of a signal cascade such that an intracellular signal is varied, such variation alters the transcription and formation of a transactivator protein encoded by a first nucleic acid construct.
  • the transactivator in turn binds to a cognate DNA segment via its DNA binding domain and activates expression of a detectable reporter.
  • the cognate DNA segment and reporter can be part of the first nucleic acid construct or a different construct.
  • activation of reporter in response to perturbations of a signal cascade is indirect or involves two steps, initially including formation of transactivator and subsequently the expression of reporter.
  • Such a system can be used, for example, to assay for agonists or antagonists that bind to a receptor.
  • the transactivator is designed to have high affinity for its cognate DNA segment which controls reporter expression.
  • the transactivator is designed to have high affinity for its cognate DNA segment which controls reporter expression.
  • the cognate sequence to which the transactivator binds is often a unique sequence in the cell.
  • the transactivator is not part of the normal cellular pathways and, as such, is not in competition with other cellular transcriptional factors for cof actors and/or transcriptional regulatory binding sites.
  • the signal amplification, achieved by the invention facilitates an analysis as to whether a particular compound affects a signal cascade, thereby increasing the sensitivity, precision and rate at which compounds can be screened to identify those capable of interacting with components of a signal cascade (e.g., binding to a receptor to generate intracellular signals).
  • Compounds identified through initial screens can serve as lead compounds for the synthesis of derivatives which can be screened for enhanced activity in subsequent rounds of screening.
  • the first nucleic acid or signal-activated transcription unit includes a first transcriptional control element that is responsive to an intracellular signal, such as one formed as a consequence of activation of a receptor.
  • This element or nucleic acid segment is operably linked to a minimal promoter that is operably linked to a nucleic acid segment that encodes the transactivator protein, which includes a DNA binding domain and a transcriptional activation domain.
  • the second nucleic acid or reporter transcription unit includes a second transcriptional control element operatively linked to a minimal promoter that is operatively linked to a nucleic acid segment encoding the reporter. Specific examples of such nucleic acid constructs are illustrated in FIGS. 2A-4C and described in Example 1 infra.
  • intracellular signal as used herein is meant to refer broadly to a component of a signal cascade, such as molecules ormacromolecules that are directly or indirectly formed as a consequence of interaction of an extracellular signal with a receptor and capable of directly or indirectly affecting the activity of the first transcriptional control element.
  • components include, but are not limited to, kinases, phosphatases, secondary messengers such as cAMP, and transcription factors that comprise a DNA binding domain and a transcriptional activation domain.
  • Specific examples of intracellular signals include, but are not limited to, activated STAT, CREB and MAP Kinase proteins.
  • Such signals can be formed directly as a consequence of an extracellular signal interacting with the receptor or can be formed indirectly as a component within a signal cascade. If a test compound is active, once it is contacted with a cell it influences the activity of the signal cascade by either interfering with or potentiating a component of the cascade. This interference or activation affects the interaction of an intracellular signal (i.e., a component of the signal cascade such as a transcription factor, for example) with the first transcriptional control element, and thus modulates the transcription of the transactivator. Such modulation affects the final signal generated since binding of transactivator to the second transcriptional control element activates transcription of the reporter that generates the detectable signal.
  • a component of the signal cascade such as a transcription factor, for example
  • Such modulation can be detected by measuring signal before and after the test cell is contacted with the test compound or with reference to a control cell treated in a similar fashion to the test cell but not contacted with the test compound.
  • a test compound is tested for its ability to bind to a receptor and trigger the formation of an intracellular signal within the cell. If the test compound is in fact a ligand for the receptor, then the intracellular signal formed interacts with the first transcriptional control element. This interaction in turn activates transcription of the nucleic acid segment encoding the transactivator.
  • the intracellular signal is a transcription factor that includes a DNA binding domain.
  • the transcription factor binds to the first transcriptional control segment via its DNA binding domain and, once bound, activates transcription of the transactivator.
  • Activation of . transcription typically is achieved through the activation domain of the transcription factor.
  • the activation domain of the transcription factor can interact with RNA polymerase already bound at the minimal promoter or facilitate polymerase binding to activate transcription of the transactivator.
  • the transactivator binds to the second transcriptional control element under appropriate conditions via its DNA binding domain. This binding activates expression of the reporter, thereby forming a detectable signal.
  • the activation domain of the transactivator can interact with the RNA polymerase to augment its activity or assist in its binding to the minimal promoter to activate transcription of the reporter.
  • Compounds that are not active because they do not interact with the receptor fail to trigger formation of the intracellular signal. Failure to form an intracellular signal means that transcription of transactivator is not activated, and thus that reporter is not expressed.
  • test compounds are screened for their ability to cause or influence signal transduction through a receptor (e.g., agonists of the receptor).
  • a receptor e.g., agonists of the receptor
  • individual cells or a population of cells expressing a receptor of interest can be placed into separate reaction sites (e.g., wells in a microtiter plate).
  • a different test compound can then be added to each reaction site.
  • Test compounds capable of transducing a signal through the receptor generate the formation of reporter, while test compounds incapable of transducing a signal do not activate the expression of reporter.
  • active compounds can be identified from the formation of a positive signal associated with reporter expression.
  • FIG. 1 A specific example of a system such as that just described is shown in FIG. 1.
  • This figure presents a schematic of how a two-step JAK/STAT signal transduction system functions.
  • a first step induced STAT phosphorylation results in binding of phosphorylated multimeric STAT proteins (i.e., an intracellular signal) to a first transcriptional control element (8X9G; an 8-fold repeat of a STAT binding sequence; see Seidel et al., Proc. Natl. Acad. Sci. USA 92:3041-3045 (1995)).
  • This interaction induces transcription of a transactivator (the tetracycline controlled transactivator (tTA), see infra) through a minimal promoter (the -CMV promoter).
  • tTA tetracycline controlled transactivator
  • induced transactivator engages a cognate DNA binding domain (seven Tet operator sequences (7XTetO)) and induces transcription through an adjacent minimal promoter (-CMV) to produce reporter (lucif erase).
  • a cognate DNA binding domain 7XTetO
  • -CMV adjacent minimal promoter
  • extracellular signal refers to any agent capable of interacting with a cell to trigger formation of an intracellular signal cascade.
  • the extracellular signal can be an ion, a molecule or macromolecule.
  • the extracellular signal binds to a receptor (see infra).
  • the extracellular signal may act directly or indirectly to generate the intracellular signal.
  • test compound refers to any agent capable of directly or indirectly triggering an intracellular signal, or directly or indirectly interacting with a component of an intracellular signal cascade to activate or interfere with the transmission of the intracellular signal.
  • the test compound can be an extracellular signal, but also includes agents that interact with a component of a signal cascade.
  • the extracellular signals and test compounds can be of a variety of general types including, but not limited to, polypeptides; carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; lipids or phospholipids; fatty acids; steroids; or amino acid analogs.
  • the compounds can be growth factors, hormones, neurotransmitters and vasodilators, for example.
  • the compounds can be of a variety of chemical types including, but not limited to, heterocyclic compounds, ⁇ -lactams, polycarbamates, oligomeric-N-substituted glycines, benzodiazepines, thiazolidinones and imidizolidinones.
  • Test compounds or extracellular signals can be obtained from libraries, such as natural product libraries or combinatorial libraries, for example.
  • the compounds can be formed on supports (e.g., polymeric beads) with or without identifying tags.
  • supports e.g., polymeric beads
  • the compounds are optionally cleaved from the support to facilitate contact with the cell and to avoid any potential steric restrictions as the cell and compound are brought into contact.
  • combinatorial libraries and methods for preparing such libraries have been described, including , for example, PCT publications WO 93/06121, WO 95/12608, WO 95/35503, WO 94/08051 and WO 95/30642, each of which is incorporated herein by reference.
  • the cells used for analysis of signal transduction should be capable of expressing the receptor of interest in functional form such that the receptor can transduce a signal to induce expression of a reporter molecule from a reporter construct.
  • the cell can endogenously express the receptor or can be genetically engineered (e.g., transfected) to express the desired receptor. If the receptor being studied is not an endogenous protein, the cells are also capable of being transfected with a nucleic acid sequence that includes the sequence for the receptor of interest, as well as the signal-activated transcription unit and the reporter transcription unit.
  • receptor is used broadly to refer to a cellular macromolecule, which interacts with an extracellular signal/compound and transduces a signal as a result of such interaction that causes, directly or indirectly, a detectable change in the transcription or translation of a gene or localization of a gene product.
  • the compound can transduce a signal alone, or may act in conjunction with another ligand. Often the signal is transmitted between the receptor and the gene by a cascade of intracellular events.
  • Many receptors are cell surface proteins that transduce a signal, directly or indirectly, to a gene. These receptors have one or more of each of the following domains: an extracellular domain to interact with a compound, a transmembrane domain and an intracellular domain.
  • Such receptors include ion channels (e.g., calcium, sodium, potassium channels), voltage-gated ion channels, ligand-gated ion channels (e.g., acetyl choline receptors, and GABA (gamma-aminobutyric acid) receptors), growth factor receptors, muscarinic receptors, glutamate receptors, adrenergic receptors, dopamine receptors (see, e.g., U.S. Pat. Nos.
  • ion channels e.g., calcium, sodium, potassium channels
  • voltage-gated ion channels e.g., acetyl choline receptors, and GABA (gamma-aminobutyric acid) receptors
  • growth factor receptors e.g., muscarinic receptors, glutamate receptors, adrenergic receptors, dopamine receptors (see, e.g., U.S. Pat. Nos.
  • the receptor is heterologous to the cell used for screening, in which case the receptor is expressed from a recombinant construct introduced into the cell.
  • Cytokine and interferon receptors are examples of cell surface receptors, some of which are part of the JAK/STAT cascade system. As described in the Background section, in this cascade system tyrosine kinases activate STAT proteins by phosphorylating them. The phosphorylated STAT proteins are capable of entering the nucleus of a cell and activating transcription by binding to SRE sites.
  • G protein coupled receptors comprise another major class of cell surface receptors and couple to effector proteins through guanine nucleotide binding regulatory proteins called G proteins.
  • G-protein-coupled receptors share a conserved structural motif including seven hydrophobic stretches of approximately 20-25 amino acids, which are surrounded by eight hydrophilic regions of variable length. It is thought that each of the seven hydrophobic regions forms a transmembrane alpha helix; the intervening hydrophilic regions are thought to form exposed loops alternately on the intracellular and extracellular side of the cellular membrane. The third hydrophilic region between transmembrane domains five and six is the intracellular domain responsible for the interaction with G proteins.
  • Binding of a ligand (e.g., a hormone) to receptor triggers the association of a G protein complex ( ⁇ , ⁇ , and ⁇ subunits) on the cytoplasmic side ofthe receptor, together with an inactive form of adenylate cyclase.
  • a G protein complex ⁇ , ⁇ , and ⁇ subunits
  • GDP is displaced from the ⁇ subunit and is replaced with GTP, the binding of which causes a conformational change that results in the dissociation ofthe ⁇ subunit from the ⁇ , and ⁇ subunits.
  • the ⁇ subunit then associates with inactive adenylate cyclase and, in so doing, activates it.
  • cAMP can then act as a second messenger in various cascades that result in the formation of active transcription factors (e.g., CREB) that activate transcription of certain genes.
  • active transcription factors e.g., CREB
  • G-protein receptors include, but are not limited to, substance K receptor, the angiotensin receptor, the ⁇ - and ⁇ -adrenergic receptors, the serotonin receptors, and PAF receptor (see, e.g., Gilman, Ann. Rev. Biochem. 56, 625-649 (1987)).
  • Some growth factor receptors such as f ⁇ broblast growth factor receptor, have tyrosine kinase activity.
  • Growth factors are polypeptides that regulate cell division and differentiation and whose function is mediated by binding to cell-surface receptors that triggers intracellular signals that alter gene expression. Growth factors bind to specific cell surface receptors and induce tyrosine phosphorylation and c-fos mRNA synthesis. Interaction between a growth factor and its cognate receptor activates protein kinase C, a family of phospholipid- and calcium-activated protein kinases.
  • Activation results in transcription of multiple protooncogene transcription factor encoding genes (e.g., c-fos, c-myc and c-jun), proteases, protease inhibitors (e.g., collagenase type I and plasminogen activator inhibitor) and adhesion molecules (e.g., intercellular adhesion molecule I).
  • protooncogene transcription factor encoding genes e.g., c-fos, c-myc and c-jun
  • proteases e.g., protease inhibitors (e.g., collagenase type I and plasminogen activator inhibitor)
  • adhesion molecules e.g., intercellular adhesion molecule I.
  • Ion channels are receptors that function as a gated pore across the cell membrane. Ion channels permit the flux of ions across the cell membrane in response to electrical or chemical gradients. The channels are classified depending upon the type of ion the passes through the channel and include, for example, calcium, sodium, and potassium. A number of different ion channels are described in U.S. Pat Nos. 5,401,629 and 5,436,128, which are incorporated herein by reference in their entirety.
  • Some ion channels are referred to as voltage-gated ion channels because they permit ion flux through the channel once a certain transmembrane potential is reached. Ion flux plays a key role in various responses including, for example, stimulus-secretion, stimulus-mitosis and stimulus contraction (see, for example, Curran, et al. (1986) Proc. Natl. Acad. Sci. USA 83:8521-8524).
  • Other ion channels are responsive to the binding of certain ligands and are referred to as ligand-gated ion channels. Examples of such channels include, for example, nicotinic acetyl choline receptors, gamma-aminobutyric acid (GABA) receptors, and excitatory amino acid receptors.
  • Some receptors are intracellular proteins, such as enzymes, nuclear receptors (e.g., FXR (Farnesoid X Receptor), PPARb (Peroxisome Proliferator Activator Receptor Delta), and RZR (Retinoid Z Receptor)), organelle receptors, and hormones. If the receptor under test is an intracellular protein, the cell type chosen should have the inherent or engineered capacity to take up the compounds of the kind being tested.
  • Both prokaryotic and eukaryotic cells can be utilized, although typically the cells are eukaryotic.
  • a number of different eukaryotic cell types can be utilized in the invention including, for example, cells from mammalian, as well as nonmammalian sources (e.g., yeast, fungus, amphibians and insects).
  • Primary cultures of natural cells (e.g., hemopoietic cells) expressing receptors of interest can also be used provided the cells naturally have, or can be transfected with, an appropriate reporter construct.
  • Suitable mammalian cells include, but are not limited to, CHO (Chinese hamster ovary) cells, HepG2 cells, BaF-3 cells, Schneider cells, COS cells (monkey kidney cells expressing SV40 T-antigen), CV-1 cells, HuTu80 cells, NTERA2 cells, NB4 cells, HL- 60 cells and HeLa cells, 293 cells (see, e.g., Graham et al., J. Gen. Virol. 36:59 (1977)), and myeloma cells like SP2 orNSO (see, e.g., Galfre and Milstein, Meth. Enzymol. 73(B):3-46 (1981)).
  • Non-mammalian eukaryotic cells can also be utilized in the present invention including, for example, insect (e.g., sp.frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha) and fungal and plant cells.
  • insect e.g., sp.frugiperda
  • yeast e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha
  • yeast e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha
  • yeast e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha
  • K. lactis K. lactis
  • H. polymorpha fungal and plant cells.
  • the assay plate can be one of the standard microtiter plates that are commercially available and well-known to those of skill in the art.
  • the assay plate can be fabricated using photolithographic techniques to form assay plates that have smaller and more numerous wells as compared to commercial microtiter plates. For example, an assay plate measuring 10 cm x 10 cm and containing 10 6 wells in an array of 1000 by 1000, each well having dimension of 100 ⁇ m x 100 ⁇ m x 100 ⁇ m (i.e., 1 nl), can be prepared using such techniques.
  • the wells themselves needmot have any particular shape.
  • Another format involves placing compounds within a matrix that allows diffusion of the compounds and cells so that they are brought into contact with one another.
  • the matrix keeps the different compounds and cells segregated.
  • One example of this approach involves suspending excess cells and a quantity of compound in a layer of soft agarose or agar (preferably low melting temperature) several mm thick.
  • cells (or test compound) are spread out on a membrane and contacted with test compound (or cells).
  • test compound or cells
  • the number of cells within each well typically ranges from 10,000 to 500,000, more typically, 50,000 to 250,000 and most typically 50,000 to 100,000.
  • the length of the time during which compounds and cells are incubated so as to allow signal transduction can be determined empirically without undue experimentation. For example, a time course study can be performed by measuring the level of transcription as a function of time. Utilizing the microtiter plate approach described above, typically reporter expression is detected about 4 to 24 hours from the time the cells and compounds were originally brought into contact with one another.
  • a reaction site e.g., a well in a microtiter plate
  • a test site e.g., a well in a microtiter plate
  • at least one of the test compounds within the reaction site is capable of activating the receptor to generate an intracellular signal.
  • Compounds located in such a reaction site can be rescreened individually or as part of a less diverse group of compounds to identify the particular compound or compounds within the original reaction site that have activity.
  • reporter expression can be t directly detected by detecting formation of transcript or of translation product using known techniques. For example, transcription product can be detected using Northern blots and the formation of certain proteins can be detected using a characteristic stain or by detecting an inherent characteristic of the protein. More typically, however, expression of reporter is determined by detecting a product formed as a consequence of an activity of the reporter. In such instances, detection of reporter expression is indirect.
  • Reporters that have an inherent characteristic that can be directly detected include GFP (green fluorescent protein). Fluorescence generated from this protein can be detected using a variety of commercially available fluorescent detection systems, including a FACS system for example.
  • the reporter is an enzyme that catalyzes the formation of a detectable product.
  • Suitable enzymes include, but are not limited to, proteases, nucleases, lipases, phosphatases, sugar hydrolases and esterases.
  • the reporter encodes an enzyme whose substrates are substantially impermeable to eukaryotic plasma membranes, thus making it possible to tightly control signal formation.
  • suitable reporter genes that encode enzymes include-, for example, CAT (chloramphenicol acetyl transferase; Alton and Vapnek (1979) Nature 282:864-869), luciferase (lux), ⁇ -galactosidase and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), each of which incorporated herein by reference.
  • Firefly luciferase is particularly suitable (see, for example, deWet (1986) Methods in Enzymology 133:3-14; deWet et al., (1985) Proc. Natl. Acad. Sci. 82:7870-7873; deWet et al. (1987) Mol. Cell. Biol. 7:725-737, each of which is incorporated by reference).
  • luciferase Four species of firefly from which the DNA encoding luciferase can be derived include: the Japanese GENJI and HEIKE fireflies, Luciola cruciata and Luciola lateralis; the East European firefly, Luciola mingrelica; and the North American firefly, Photinus pyralis (commercially available from Promega as the plasmid pGEM).
  • the glow-worm Lampyris noctiluca is a further source of luciferase, having 84% sequence identity to that of Photinus pyralis.
  • the reporter is part of a cascade.
  • the reporter can activate the expression of a second reporter, which can activate yet another reporter, and so on.
  • Such reporter schemes have been described, for example, in PCT publication WO 98/25146, which is incorporated herein y reference.
  • Control assays can be conducted in parallel with the test assays to provide greater certainty in the results.
  • One type of control involves using cells similar to the test cells, except the cells do not express the receptor of interest.
  • the control cell is contacted with test compound under conditions that are identical to those used with the test cell. Ii expression of reporter is higher for the test cell as compared to the control cell, this result indicates that the test compound has authentic signal transduction activity.
  • the control cell is identical to the test cell, but the control cell is not contacted with the test compound. Again, increased expression of reporter in the control experiment relative to the test experiment indicates that the test compound has bona fide activity.
  • certain methods of the invention can be utilized to screen for antagonists of receptors of interest.
  • the amplified signal generated using the methods of the present invention can be used in competition experiments to more readily detect diminution in signal resulting from antagonist binding.
  • Methods for screening for antagonist binding involve two separate trials.
  • a compound known to activate a receptor of interest e.g., an agonist
  • the cell contains a signal-activated transcription unit as described above that includes a first transcriptional control element that is responsive to an intracellular signal generated by the receptor, as well as a reporter transcription unit.
  • interaction of the compound with the receptor results in transcription of transactivator and subsequent expression of reporter that can be detected as a positive signal.
  • the second trial is conducted using an identical cell under identical conditions to the first trial, except that the cell is also contacted with test compound that is a potential antagonist. Expression of reporter in the second cell is also detected and compared with the level of expression in the first trial. Decreased expression in the second trial indicates that the test compound is an antagonist.
  • the basic components of the signal-activated nucleic acid comprises a first transcriptional control element in operable linkage with a promoter which in turn is operably linked to a sequence encoding for a transactivator protein.
  • the first transcriptional control element is operatively linked upstream (i.e., 5') of a promoter (typically a minimal promoter - see infra) through a phosphodiester bond at a suitable distance to allow for transcription of a downstream segment encoding the transactivator protein following interaction of an intracellular signal with the first transcriptional regulatory element.
  • the distance between the first transcriptional regulatory sequence and the promoter can vary from about 10 bases to several kilobases. Generally, however, the distance between these two elements is about 200-400 base pairs.
  • the distance between the promoter and segment encoding the transactivator can also vary, although typically these elements are separated by approximately 30-100 base pairs.
  • the first transcriptional control element is any nucleotide sequence that is responsive to an intracellular signal.
  • responsive when used in reference to the first transcriptional control element is meant the ability of the transcriptional control element to interact (e.g., bind) with an intracellular signal formed upon activation of the receptor and to activate transcription of the segment encoding the transactivator protein.
  • the first transcriptional control element is one capable of binding to a protein activated through a signal cascade process.
  • the first transcriptional control element includes one or more STAT response element sequences to which an activated STAT protein can bind (see, e.g., Seidel , M., et al., Proc. Natl. Acad. Sci. USA, 92: 3041-3045 (1995); Darnell Jr., J.E., Science, 277:1630-1635 (1997); and Lamb, P., et al., Drug Discovery Today 3:122-130, (1998), each of which are incorporated by reference in their entirety).
  • the first transcriptional control element includes a CRE sequence (see, e.g., Stables, J., et al., J. Receptor and Signal Transduction Research 19:395-410 (1999), which is incorporated by reference in its entirety).
  • the first transcriptional control sequence includes a serum response element.
  • the response element is Gal4 (see Example 1, supra, and FIGS. 3 A and 3B).
  • the nucleic acid segment that encodes for the transactivator protein can be any sequence that encodes for a protein having a DNA binding domain and a transcriptional activation domain, provided the DNA binding domain can bind to the selected second transcriptional control element and the activation domain is capable of activating expression of reporter.
  • the segment encodes for a fusion protein, wherein the DNA binding domain and the transcriptional activator domain are from different proteins.
  • both domains may be from prokaryotic or eukaryotic proteins or one domain may be from a prokaryotic domain while the other domain is an eukaryotic domain.
  • the nucleic acid segment does not encode a fusion protein, but instead encodes a naturally occurring protein that includes both a DNA binding domain and a transcriptional activation domain (e.g., Gal4).
  • the sequence may also include a nucleic acid segment encoding for a linker polypeptide that connects the two domains.
  • the DNA binding domain can be a prokaryotic or an eukaryotic nucleotide sequence that encodes a polypeptide capable of binding to a DNA sequence, specifically the cognate second transcriptional control element of the reporter transcription unit.
  • a prokaryotic DNA binding domain to be utilized in eukaryotic cells and an eukaryotic DNA binding domain to be utilized in prokaryotic cells.
  • DNA binding domains examples include tet repressors, mutated tet repressors, the DNA binding domain of bacterial lex A protein, the DNA binding domain of Gal4, the DNA binding domain of STAT proteins and CREB.
  • the tet repressor sequences can be either wild type or mutant types. Wild type tet repressors refer to tet repressor proteins that are naturally-occurring and which repress transcription from a tet operator sequences in the absence of tetracycline.
  • a mutated tet repressor in contrast, refers to polypeptides having an amino acid sequence which is similar to the wild-type tet repressor, but which have at least one amino acid difference from the wild-type repressor.
  • a mutated tet repressor can bind to a tet operator sequence and hence retains the DNA binding specificity of a wild-type tet repressor.
  • tet repressor sequences there are a wide variety of different types of tet repressor sequences that can be utilized with the invention, including, for example, classes A, B, C, D and E.
  • the nucleotide and amino acid sequences of such tet repressors and methods for obtaining such sequences are described, for example, in Waters, S.H. et al. (1983) Nucl. Acids, Res 11:6089- 6105; Hillen, W. et al. (1983) Nucl. Acids Res. 11:525-539; Postle, K. (1984) Nucl. Acids Res. 12:4849-4863; Unger, B. et al. (1984) Gene 31:103-108; Unger, B.
  • Mutated tet repressors can be prepared using standard molecular biology techniques, including, for example, subjecting a nucleic acid encoding a wild-type Tet repressor to random mutagenesis or introducing a nucleotide change into a nucleic acid encoding wild-type tet repressor by site directed mutagenesis or PCR-mediated mutagenesis using nucleic acid primers incorporating the nucleotide mutation(s).
  • Other methods including mutating an already mutant form of tet repressor, is described in U.S. Pat. Nos. 5,589,362; 5,654,168; and 5,912,411, each of which is incorporated by reference in its entirety.
  • the Gal4 DNA binding domain has been well characterized.
  • the DNA binding domain of the yeast Gal4 protein comprises at least the first 74 amino acids thereof (see, for example, Keegan et al., (1986) Science 231:699-704).
  • the first 90 or more amino acids of the Gal4 protein are used, and in still other instances, at least the first 147 amino acid residues of Gal4 protein are utilized as the DNA binding domain. Cloning of this binding domain is described, for example, by Umesono et al. (1991) Cell 65:1255-1266. 3. Segment Encoding Activation Domain
  • the nucleotide segment encoding the transcriptional activation domain of the transactivator protein is operably linked to the nucleotide segment encoding the DNA binding domain of the transactivator.
  • the transcriptional activation domain is selected to be appropriate for the type of cell utilized. For example, typically an eukaryotic activation domain is used with eukaryotic cells and a prokaryotic activation domain is used with prokaryotic cells.
  • the activation domain is capable of activating transcription on its own. In other instances, however, the activation domain activates transcription by an indirect mechanism, such as by recruiting a transcriptional activation protein to interact with the transactivator protein.
  • the transcriptional activation domain includes polypeptides that are capable of directly or indirectly activating transcription.
  • a number of polypeptides that directly function to activate transcription in eukaryotic cells are known in the art. For example, numerous transcriptional activation domains of many DNA binding proteins have been described and shown to retain activation activity even when the activation domain is linked to a heterologous protein. Transcriptional activation domains have been grouped into categories based upon certain structural similarities. Examples, of such categories include: acidic transcription activation domains, proline-rich activation domains, serine/threonme-rich transcription activation domains and glutamine-rich transcription activation domains.
  • acidic transcription activation domains include the herpes simplex virus virion protein 16 (referred to herein simply as VP16; see below), and residues 753-881 of Gal4.
  • proline-rich activation domains include amino acid residues 399-499 of CTF/NF1 (Mermod et al. (1989) Cell 58:741-753) and amino acid residues 31-76 of AP2.
  • serine/threonine-rich activation domains include residues 175-269 of Octl and amino acid residues 132-243 of Spl (Courey and Tjian (1988) Cell 55:867-898).
  • the amino acid sequences of the foregoing activation domains are also described, for example, by Seidel, K.
  • VP16 is one specific example of a suitable transcriptional activation domain.
  • the amino acid sequence of the protein and methods for obtaining the nucleotide sequence encoding the polypeptide has been describe by Triezenberg, et al. (1988) Genes Dev. 2:718-729). Suitable residues from the C-terminal end of VP16 are disclosed, for example, in U.S. Pat. Nos. 5,464,758; 5,589,362; and Seidel, K. et al. (1992) EMBO J. 13:4961-4968, each of which incorporated by reference in its entirety.
  • the activation domain comprises the C-terminal 130 amino acids of VP 16.
  • Other specific activation domains that are suitable for the present invention include the Gal4 activation domain, the STAT protein activation domain and the Elk-1 activation domain.
  • Novel transcriptional activation domains can be readily identified utilizing known methods and are within the scope of the present invention.
  • transcriptional activation activity of a polypeptide can be assayed by preparing a fusion protein by linking a polypeptide to a polypeptide known to have DNA binding activity. The amount of expression of a target segment that includes a transcriptional regulatory segment which is capable of binding to the DNA binding domain of the fusion protein is then determined.
  • Seidel, K et al. (1992) EMBO J. 11:4961-4968 (and references cited therein) describes a system that utilizes a fusion protein comprising a Gal4 DNA binding domain (e.g., residues 1-93) operably linked to a putative transcriptional activation domain.
  • Active transcriptional activation domains can be identified by their ability to activate transcription of a reporter gene in operable linkage to Gal4 DNA binding sites.
  • the nucleotide sequences encoding these two domains are generally ligated to each other in-frame to create a chimeric gene encoding a fusion protein.
  • Linkage can be accomplished according to conventional techniques including, for example, blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and ligation with appropriate ligases.
  • the two nucleotide sequences can be operatively linked by any other means that preserve the function of each polypeptide domain (e.g., chemical crosslinking).
  • a segment encoding a naturally occurring protein is used rather than a fusion protein (e.g., Gal4).
  • the full-length sequence encoding the protein can be used and ligation of two nucleotide sequences is unnecessary, unless it is desired to insert a linking sequence between the two domains.
  • the activation domain is matched with cell type, i.e., a eukaryotic domain for a eukaryotic cell and a prokaryotic domain for a prokaryotic cell.
  • a promoter is typically operably linked to the segment that encodes the transactivator protein.
  • the promoter is a minimal promoter.
  • a minimal promoter is a partial promoter sequence that defines the start site of transcription for the linked sequence to be transcribed.
  • a minimal promoter is unable to initiate transcription efficiently on its own, if at all.
  • a minimal promoter is unable to activate transcription until a transcriptional activator binds to an operably linked transcriptional control site (e.g., binding of an activator such as a STAT or CREB protein to the first transcriptional control element) and activates transcription via its activation domain.
  • Other suitable minimal promoters include, for example, those listed in U.S. Pat. No. 5,859,310. Additional suitable minimal promoters can be identified utilizing standard molecular biology techniques. For instance, a functional promoter that activates transcription of a contiguously linked reporter gene (see below) can be progressively deleted using standard techniques until it no longer activates transcription of the reporter by itself but requires the presence of an additional regulatory element (e.g., transcriptional activation domain).
  • minimal promoters that can be prepared and utilized in yeast include, for example, those prepared from GALl-10 (Johnson and Davies (1984) Mol. Cell Biol. 4:1440-1448), ADH2 (Russell et al. (1983) J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), MF ⁇ (Herskowitz and Oshima (1982) in The Molecular Biology ofthe Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, N. Y., pp.
  • minimal promoters that can be prepared and utilized in mammalian systems, include, for example those prepared from SV40 promoter (de laLuma, et ⁇ /.,(1998) Gene 62:121), RSV promoter (Yates, et al, (1985) Nature 313:812), and MMTV promoter (Lee, et ⁇ /.,(1981) Nature 294:228).
  • a convenient promoter for use in insects include the baculoviras Autographa Calif ornica nuclear polyhedrosis virus (NcMNPV) (Kitts, et al, (1993) Nucleic Acids Research 18:5667). V. Reporter Transcription Unit
  • the second transcription unit typically comprises (in the 5' to 3' direction) a second transcriptional control element segment that is responsive to the activation domain of the transactivator protein, a promoter (typically a minimal promoter) and a segment encoding a reporter.
  • a promoter typically a minimal promoter
  • the second transcriptional control element is operatively linked upstream (i.e., 5') at a sufficient distance to allow for transcription of the downstream reporter segment once the transcriptional activator protein binds to the second transcriptional regulatory element. Similar to the orientation in the first transcriptional unit, the distance between the second transcriptional regulatory sequence and the minimal promoter can vary.
  • the distance between these two elements is about 200-400 base pairs.
  • the promoter and the segment encoding the reporter are also operably linked; the distance between the promoter and sequence encoding the reporter can also vary, although typically these elements are separated by approximately 30 -100 base pairs.
  • activation of the reporter segment typically is under the direct control of the transactivator protein. In other instances, however, the activation domain does not directly activate transcription of the reporter but instead recruits other activators that activate transcription.
  • the second transcriptional control element is selected to be a cognate DNA sequence that specifically binds to the DNA binding domain of the transactivator protein.
  • the transcriptional control element exists as a single sequence. More frequently, however, the sequence is present as multiple repeats (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats). A greater number of regulatory segments can provide for greater control in the regulation of transcription of the reporter sequence.
  • the tet operator is one example of a suitable second transcriptional control element when the tet repressor comprises the DNA binding domain of the transactivator protein.
  • the tet operator sequence can be obtained, for example, according to methods described by Hillen and Wissmann, "Topics in Molecular and Structural Biology," in Protein- Nucleic Acid Interaction, Saeger and Heinemann, eds., Macmillan, London, 1989, vol. 10, pp. 143-162, which is incorporated herein by reference.
  • Other Tet operator sequences that are useful in the present invention are described by Waters, et al, Nucl. Acids, Res. 11:6089- 6105 (1983); Postle et al., Nucl. Acids Res.
  • tet operator is meant to encompasses al the different classes of Tet operators, including, for example, class A, B, C, D and E.
  • Nucleotide sequences for these classes are described, for example, by Waters, et al. (1983) Nucl. Acids Res 11:6089-6105; Hillen, W. and SchoUenmeier, K. (1983) Nucl. Acids Res. 11:525-539; Stuber D. and Bujard, H. (1981) Proc. Natl. Acad. Sci. USA 78:167-171; Unger, B. et al. (1984) Gene 31:103-108; Unger, B. et al. Nucl, Acids Res. 12:7693-7703 (1984); Tovar, K. et al. Mol. Gen. Genet. 215:76-80 (1988), each of which is incorporated by reference.
  • tet operators are described in U.S. Pat. Nos. 5,589,362, which is also incorporated by reference.
  • the number of tet operators can vary. In some instances, a single operator is used. More typically, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more operators are employed.
  • Gal4 response elements are appropriate sequences for the second transcriptional control element when a Gal4 DNA binding domain comprises part of the transactivator protein.
  • Exemplary Gal4 response elements include certain palindromic 17- mers including, for example, 17 MX described by Webster et al., (Cell 52: 169-178 (1988), incorporated by reference in its entirety) as well as derivatives thereof. Additional examples of suitable Gal4 response elements include, for example, those described by Hollenberg and Evans (Cell 55:899-906 (1988)) and Webster et al. (Cell 54:199-207 (1988)) and in U.S. Pat. Nos. 5,906,920, each of which is incorporated herein by reference in its entirety.
  • a cognate response element to the STAT DNA binding domain is used if the
  • DNA binding domain of a STAT protein comprises the DNA binding domain of the transactivator protein.
  • Suitable segments include any of a number of different SRE (STAT response elements) which include, but are not limited to, the 9G and repeats of a SRE segment, such as the 8X9G sequence (i.e., the 9G sequence repeated 8 times; see, e.g., Seidel, M., et al., Proc. Natl. Acad. Sci. USA, 92:3041-3045 (1995)).
  • SRE STAT response elements
  • a reporter sequence includes any gene that expresses a detectable gene product, which may be RNA or protein. Examples of suitable reporters are described above in the discussion of the detecting steps of the method.
  • the reporter sequence is operably linked to a minimal promoter which is operably linked to a second transcriptional control element. This basic construct can be repeated one or more times to achieve further amplification of signal. If present as a repeat, the repeating units are typically separated by a spacer of sufficient length to reduce the frequency of inter unit site-specific recombination which could reduce signal.
  • a sufficient space is at least 100-250 nucleotides of non-interfering sequence, and can be readily determined empirically by varying the length of the spacer and determining optimal lengths for maximal gene expression.
  • the promoter that is operably linked to the reporter typically is a minimal promoter. Suitable promoters include those described above for the signal-activated transcription unit, especially CMV minimal promoters.
  • nucleicacids that include the signal-activated transcription unit and/or the reporter transcription unit
  • plasmids including pSV2, pBC12BL p91023, pCDNA series, pCMVl, and pMAMneo
  • lytic virus vectors e.g., vaccinia virus, adenovirus
  • episomal virus vectors e.g., bovine papillomavirus
  • retroviral vectors e.g., murine retroviruses
  • suitable vectors include the plasmids pYepSecl (Baldari, et al., (1987) EMBO J. 6:229- 234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123) and PYES2 (available from Invitrogen Corp., San Diego, CA), as well as Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) such as the pYES series and pGPD-2, for example.
  • Yeast Integrating plasmids e.g., YIp5
  • Yeast Replicating plasmids the YRp series plasmids
  • the YRp series plasmids such as the pYES series and p
  • Suitable vectors for insect cells include a variety of baculovirus vectors, including, but not limited to, pFastBacl, pFastBacHT series, pBluesBac4.5, pBluesBacHis series, the pMelBac series, the pVL series (e.g., pVL1392/1393; see, e.g., Lucklow, V.A., and Summers, M.D. (1989) Virology 170:31- 39) and the pAc series (Smith, et al, (1983) Mol. Cell Biol.: 3:2156-2165). Suitable insect viruses are further described by O'Reilly et al.
  • Vectors for use in bacteria include, for example, pBR322 derived vectors, such as pBLUESCRIFTTM, pUC18/19, as well as phage derived vectors.
  • plasmids containing the transcription units described above (or individual elements thereof) utilizes known ligation techniques.
  • isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required.
  • the plasmids can be analyzed by standard techniques, such as by restriction endonuclease digestion and/or sequencing according to known methods.
  • Methods for constructing suitable vectors are described by e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, CA (Berger); and Current Protocols in Molecular Biology, F.M.
  • the nucleic acid(s) containing the signal-activated transcription sequence and the receptor transcription sequence can be introduced into a host cell using standard molecular biological techniques for transfecting eukaryotic cells, such as those described by Sambrook, et al. in Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press (1989), which is incorporated by reference in its entirety. Examples of such techniques include, for example, calcium phosphate co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation and microinjection.
  • a gene that contains a selectable marker (e.g., drug resistance) is generally introduced into the host cells along with the nucleic acid of interest.
  • selectable markers e.g., drug resistance
  • Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, G418, tetracycline and hygromycin.
  • selectable markers may encode proteins that complement auxotrophic deficiencies or supply critical nutrients not available from complex media. Numerous other selectable markers are known in the art, some of which are described for instance in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1984, which is incorporated by reference in its entirety.
  • Selectable markers can be introduced on a separate plasmid from the nucleic acids of interest or introduced on the same plasmid. Host cells transfected with a nucleic acid of interest and a gene for a selectable marker can be identified by selecting for cells having the selectable marker.
  • the signal-activated transcription unit and reporter transcription unit can be included on separate plasmids or included on the same plasmid.
  • the an exogenous reporter can be included on a separate plasmid or a plasmid including the signal-activated transcription unit and/or reporter transcription unit.
  • a number of different assay systems can be prepared.
  • a variety of assay methods can be based upon systems that utilize a transactivator that is a fusion protein of the prokaryotic tet repressor operatively linked to the eukaryotic transcriptional activation domain VP16.
  • a transactivator that is a fusion protein of the prokaryotic tet repressor operatively linked to the eukaryotic transcriptional activation domain VP16.
  • tTA tetracycline controlled transactivator
  • the cognate sequence in the second transcriptional control unit is a tet operator, typically multiple tet operator sequences, operably linked to a minimal CMV promoter, which in turn is operably linked to a reporter gene.
  • tet repressor In the presence of tetracycline or tetracycline analog, tet repressor does not bind to its cognate sequence, tet operator. Thus, in certain methods of the invention utilizing this transactivator, cells are incubated in the absence of tetracycline or a tetracycline analog prior to detection of reporter expression, as binding of the tet repressor of the transactivator protein is required for reporter expression.
  • gene (i.e., reporter) regulation in the systems of the present invention depends upon an alteration of the activity of a signal cascade component, such as a ligand induced activation of a receptor.
  • a signal cascade component such as a ligand induced activation of a receptor.
  • tet or tet analog concentrations can be altered during an assay, this type of control is unnecessary as the amount of signal amplification has been found to be the same whether or not cells are initially contacted with tet.
  • a transactivator system utilizing tTA can be used for assaying a variety of different signal transduction systems.
  • a tTA transactivator can be used in a CRE/CREB system in ' which the receptor transduces an extracellular signal to activate the cascade system discussed in the Background section to form an activated CREB protein that binds to a CRE site on the first transcription unit.
  • the signal-activated transcription unit in this instance includes a CRE binding element (the first transcriptional control element), a minimal promoter, (e.g., a minimal TK promoter) and a nucleic acid segment encoding tTA.
  • the second transcriptional control element on the reporter transcription unit includes one or more tet operator sequences. Additional detail regarding such a system is set forth in Examples 1 and 2 and FIGS. 4A and 4B.
  • a tTA transactivator can be utilized with a JAK/STAT system in which interaction of a compound with a suitable receptor activates the signal cascade that results in the formation of an activated STAT protein.
  • the first transcriptional control element in the signal-activated transcription unit is a SRE nucleotide segment to which the STAT protein binds.
  • Other aspects of the system are as described for the CRE/CREB system and are described in fuller detail in Examples 1 and 2 and illustrated in FIG. 1.
  • tTA-based systems can be slightly modified by using a mutated tet repressor that binds to a tet operator in the presence of tet or tet analog, instead of the absence of tet or tet analog.
  • a mutated tet repressor that binds to a tet operator in the presence of tet or tet analog, instead of the absence of tet or tet analog.
  • the cells are incubated with tet or tet analog prior to detection of reporter expression.
  • a variety of related systems can be similarly developed by varying, for example, one or more components of the transactivator protein.
  • a tet repressor can be paired with a Gal4 transcriptional activation domain or a STAT transcriptional activation domain.
  • the second transcriptional control element is a tet operator to which the DNA binding domain of the tet repressor can bind.
  • Both systems can be utilized in a CRE/CREB, JAK/STAT system, MAP-K/Elk-1, or other signal transduction systems. In other systems, both the binding and transactivator domains are from the
  • Gal4 protein or the STAT protein.
  • FIG. 4C an example of one suitable Gal4 plasmid construct is shown in FIG. 4C.
  • the construct claim in this figure is essentially the same as the two-step construct, pTR Lux:8X9G, described in FIG. 2B, except the Tta transactivator is replaced with the Gal4 transactivator (see, e.g., Lawler, J. F. et al., Anal Biochem 269, 133- 138 (1999)) and the 7XTet by the Gal4 DNA binding domain from pFR Luc (Stratagene).
  • Other configurations can readily be utilized by selecting from the various elements listed above.
  • the second transcriptional control element of the second transcription control unit include a cognate sequence for the DNA binding domain of the transactivator protein
  • the transcriptional activation domain of the transactivator protein be a eukaryotic activation domain if the cell is an eukaryotic cell and a prokaryotic activation domain if the cell is a prokaryotic cell.
  • the DNA binding domain is selected to be a prokaryotic domain for eukaryotic cells and selected to be a eukaryotic domain for prokaryotic cells. As described above, this reduces competition between the transactivator and other transcription factors for the cognate binding segment on the reporter transcription unit (i.e., the second transcriptional control element).
  • analog compounds identified using the methods of the invention as being capable of activating signal transduction through a receptor of interest can serve as lead compounds for the synthesis of analog compounds.
  • the analogs should have a stabilized electronic configuration and molecular conformation that allows key functional groups to be presented to the receptor in substantially the same way as the lead compound.
  • the analog compounds typically have spatial electronic properties which are comparable to the binding region, although the molecules can be smaller molecules than the lead compound (e.g., frequently having a molecular weight below about 2 kD and preferably below about 1 kD).
  • Identification of analog compounds can be performed through use of techniques such as self- consistent field (SCF) analysis, configuration interaction (Cl) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are available. See, e.g., Rein et al., Computer- Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York, 1989).
  • analogs Once analogs have been prepared, they can be screened using the methods of the invention to identify those analogs that have the anticipated activity. Such compounds can then be subjected to further analysis to identify those compounds that appear to have the greatest potential as pharmaceutical agents. Alternatively, analogs shown to have activity through the screening methods of the invention can serve as lead compounds in the preparation of still further analogs, which can be screened by the methods of the invention. The cycle of screening, synthesizing analogs and rescreening can be repeated multiple times.
  • Transducing compounds identified by the above methods or analogs thereof can be formulated for therapeutic use as pharmaceutical compositions.
  • Such compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, usually sterile, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Further, guidance regarding the development of pharmaceutical compounds, their formulation and delivery, is set forth, for example, in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985), and Langer, Science 249:1527-1533 (1990).
  • the signal-activated transcription unit and reporter transcription unit are heterologous nucleic acids that are introduced into the cell through standard molecular biological techniques. These nucleic acids can be separate nucleic acids or part of the same nucleic acid. In some instances, these transcription units or elements thereof can be introduced into a cell such that the sequences become integrated within the chromosome of the cell. Using such techniques, it is possible to develop transgenic animals having the introduced sequences.
  • the transcription units or elements thereof can be integrated into the chromosome of a eukaryotic cell or animal according to known methods.
  • nucleic acids containing the element(s) to be integrated are prepared in which the segment to be integrated is flanked at its 5' and 3' ends by additional nucleic acid which can undergo homologous recombination with a chromosome of a cell or organism.
  • the additional flanking segments need to be of sufficient length to allow for successful recombination.
  • integration of desired segments can be integrated into a predetermined location on a target nucleic acid utilizing enzyme-assisted site-specific integration systems.
  • enzyme-assisted site-specific integration systems include the Cre recombinase-lox target system (see, e.g., Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad. Sci.
  • nucleic acid segment encoding a minimal promoter and the transactivator is prepared that has sufficiently long flanking nucleic acid segments that allow for homologous recombination within the chromosome of an eukaryotic cell or organism.
  • the segment is incorporated to be operably linked to a transcriptional control element within the chromosome.
  • the entire signal-activated transcription unit and/or the reporter transcription unit (or various elements thereof) can be similarly integrated. Detection of reporter expression in such instances would typically utilize various whole body imaging techniques that are known in the art.
  • Kits The invention also provides kits for conducting the methods of the invention.
  • the kit includes a nucleic acid that includes the signal-activated transcription unit (i.e., first transcriptional control element operably linked to a segment encoding a transactivator protein) and a second nucleic acid that includes the reporter activated transcription unit (i.e., a second transcriptional control element and a segment encoding the reporter).
  • the first transcriptional control element is selected to be responsive. to an intracellular signal generated by a receptor of interest.
  • the second transcriptional control element is selected to be capable of interacting with the DNA binding domain of the transactivator.
  • the various elements can be chosen from the various elements described above.
  • the kits also include a third nucleic acid that encodes for the receptor of interest. The identity of the various elements can be as described above.
  • the transactivator includes a mutated tet repressor which only binds to the tet operator in the presence of tetracycline or tetracycline analog
  • the kit can also include a quantity of tetracycline or an analog thereof.
  • FIGS. 4A and 4B Plasmid constructs for certain CRE/CREB systems are illustrated in FIGS. 4A and 4B. These plasmids respond to CREB activation through a CRE transcriptional control element (6CRE).
  • the 6 CRE and -TK promoter elements are from the plasmid 6CRE-Luc Hyg (Stables, J., et al. (1999) J. Receptor and Signal Transduction Research, 19: 395-410).
  • the Tetracycline Transactivator (Tta), the 7XtetO and -CMV promoter elements are from plasmids described by Bujard (Gossen, M., and Bujard, H., Pro. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • the Sr ⁇ promoter is derived from plasmid pcDL-Sr 296 (see, e.g., Yutaka Takebe et al, Mol. Cell. Biol., 8:466-472 (1998)).
  • the PuroR gene from pPUR (Clontech Laboratories) provides for puromycin selection in mammalian cells. Luciferase gene is from Photinus.
  • the Stop transcription termination cassette sequences are derived from pBS302 (Life Technologies) by deletion of the LoxP sites.
  • plasmids are exemplary of systems that respond to STAT activation through the 8X9G binding domain, an eight fold repeat of the sequence 5'- tgcatattcctggaagtctgca-3' (modified from the Ly-6E GAS sequence described by H. Martin Seidel et al., Proc. Natl ' Acad. Sci. USA 92:3041-3045 (1995), which is incorporated by reference in its entirety).
  • MAP-K/Elk-1 plasmid constructs are illustrated in FIGS. 3A and 3B. These constructs respond to Gal4 activation.
  • the Gal4-Elkl expression cassette is from the plasmid ⁇ SG/Gal4/Elk-l described in Stephen Rees et al, Neuroreport 9:2703-2708 (1998).
  • the Gal4 DNA binding domain is from the plasmid, pFR-Luc (Stratagene).
  • a variety of cells were used to demonstrate the efficacy of the assay, including HepG2, CHO and BaF3.
  • Cells were transfected with linearized plasmid DNAs by electroporation. Plasmid DNA was linearized through either Seal or Fspl restriction enzyme digestion. These sites are contained within the Ampicillin resistance gene and do not disrupt the genes involved in eukaryotic expression. Electroporation was performed by suspension of 10 7 cells in 0.8 ml serum free RPMI with 40 ⁇ g linearized DNA at 1 mg/ml in water. The cells were chilled on ice for 10 minutes. The cell/DNA mix was then transferred into a chilled 0.4 cm gap cuvette and pulsed at 400V/250 ⁇ F.
  • HepG2 Cells were prepared the day prior to the assay by seeding wells of a 96 well plate with 50,000 cells per well in 100 ⁇ l DMEM/F-12 with 1%FBS and no phenol red and incubated at 37 °C for 16 hrs. Ligands were added in 5 ⁇ l to the 100 ⁇ l assay volume as 20X concentrates in culture medium and the assay incubated at 37 °C for 6 hours. Luciferase activity was measured using the Luclite kit (Packard) and counted on a TopCount (Packard). When tetracycline was present in the assay, it was added when the plates were seeded.
  • Cells were prepared the day prior to the assay by seeding wells of a 96 well plate with 100,000 cells per well in 100 ⁇ l DMEM/F-12 without FBS/phenol red and incubated at 37 °C for 16 hrs. Ligands were added in 5 ⁇ l to the 100 ⁇ l assay volume as 20X concentrates in culture medium and the assay was incubated at 37 °C for 6 hours. Luciferase activity was measured using the Luclite kit (Packard) and counted on a TopCount (Packard).
  • BaF3 Cells Cells were prepared the day prior to the assay by seeding wells of a 96 well plate with 100,000 cells per well in 100 ⁇ l RPMI without WeHi or phenol red and incubated at 37 °C for 16 hrs. Ligands were added in 5 ⁇ l to the 100 ⁇ l assay volume as 20X concentrates in culture medium and the assay was incubated at 37 °C for 6 hours. Luciferase activity was measured using the Luclite kit (Packard) and counted on a TopCount (Packard). When tetracycline was present in the assay, it was added when the plates were seeded.
  • FIGS. 5A and 5B show dose response curves for reporter cells with both these cytokines using cells harboring the one-step construct (i.e., the pEWP-LuxRE:8X96 construct; FIG. 2A) and the two-step construct (i.e., the pTRLuxRE:8X9G construct; FIG. 2B).
  • OSM Oncostatin M
  • IFN ⁇ Gamma Interferon
  • reporter expression resulted from the binding of activated STAT proteins to the 8X9G transcriptional control element. Such binding triggers the transcription of tTA which binds to the tet operator to activate transcription of reporter.
  • Comparable EC50s for both ligands result for both types of constructs (note the different Y axis scales).
  • the magnitude of the responses to these cytokines was much greater in the pTR Lux RE:8X9G two-step construct cells than in the pEWP-Lux RE:8X9G one-step cells, but the potencies and therefore the sensitivities of these cytokines to detection in the assay is equivalent.
  • the magnitude of the signal for the two step construct system was approximately 20-fold higher than the single-step construct.
  • the fold induction of measured luciferase activity i.e. , the ratio of signal intensities between induced signal (i.e., ligand addition) and basal signal (no ligand)
  • induced signal i.e., ligand addition
  • basal signal no ligand
  • 5D plots the effect of tetracycline in the assay medium and is consistent with the inhibitory effect of tetracycline on Tta transactivation previously shown by Bujard (Gosen, M., et al. (1995) Science 268:1766-1769.).
  • OSM Oncostatin M
  • BaF3 cells are cytokine dependent cells that are used extensively in the analysis of cytokine mediated signal trarisduction (see, e.g., Klutcher et al., Blood 91:3927-34 (1991); Tago, K. et al., J Biochem (Tokio) 123:659-67 (1998); Suzuli, J. et al., Oncogene 2:1689-97 (1997); Ghilargy, N. et al., Mol Endocrinol 11:393-99 (1997); Nandurkar, N.H. et al., Oncogene 12:585-93 (1996); and Sakamaki, K.
  • FIGS. 6 A and 6B illustrate another example of the differential magnitude of response when cells were contacted with (10 ng/ml) JJ -3 (note in FIG. 6A, the Y-axis is a linear scale; in FIG. 6B, the Y-axis is a loglO scale to show the single-step response).
  • the IL-3 cytokine response was qualitatively similar to the observed cytokine responses in HepG2 cells. In this case, however, the magnitude of the pEWP-Lux RE:8X9G response (i.e., the one-step system) was much lower than that obtained by the Two-step construct, pTR Lux RE:8X9G. Unlike the HepG2 cells, the single-step response of IL-3 was only 3-fold, whereas the two-step response was 11-fold. This indicates that using the two-step assay enables one to detect signaling events within BaF3 cells that might not otherwise be detected.
  • Tetracycline regulation i.e., assays conducted in presence of 1 ⁇ g/ml
  • cytokines for BaF3 cells harboring the two-step construct pTRLuxRE:8X9G is shown in FIG. 6C.
  • BaF3 cells transfected with the human thrombopoietin (TPO) receptor showed repressed signal from a TPO mimetic peptide, 705, as well as a suppressed IL-3 cytokine response from the endogenous JJL-3 receptor when cells were contacted with JJL-3, as expected for a system using tTA and the tet operator (see, Cwirla et al. Science 276:1696- 1699 (1997)).
  • Results without tetracycline are indicated with a minus sign; results with tetracycline are indicated with a plus sign.
  • Stable CHO cell lines have been established with a wide variety of cell surface receptors.
  • the CHO cell line used for these assays express the human Insulin receptor Reporter expression was under tTA transactivator and tet operator control (see FIGS. 3 A and 3B).
  • Insulin receptors are known to trigger MAP Kinase mediated signal transduction (see, Yamauchi, K. et al., J. Biol Chem. 268:14597-600 (1993)).
  • the dose response results in FIGS. 7A show the same type of responses seen in the BaF3 and HepG2 cells systems using the Jak-STAT reporter system. As shown in FIG.
  • CHO cells transfected with the CCKb receptor were utilized to create the cell lines used for measuring CREB mediated signal transduction (see FIGS. 4A and 4B)
  • this system also used tTA as transactivator and the tet operator as the control element regulating reporter formation. Phosphorylation of CREB results from elevations in cAMP levels within the cell after stimulation of the CCKb receptor by CCK8 (see, e.g., Detgen, K. et al., Am. /. Physiol. 293:61449-57 (1997)).
  • FIGS. 8A-8B show results for the effect of tetracycline on signal generation (one-step and two-step, respectively);
  • FIGS. 8C and 8D are dose response curves for titrations with CCK8 (one-step and two-step respectively).
  • Tetracycline had no effect upon signaling in the one-step system but repressed signal generation 21-fold in the two-step system.
  • There was significant enhancement in overall signal for the two-step system again consistent with the differential responses obtained with the other cell types and signal transduction assays.
  • the fold induction for the one-step was approximately lOx for the one-step system and 7x for the two-step system.
  • the increase in signal for the two-step system relative to the one-step system was approximately 38-fold, demonstrating again the significant signal enhancement that can be achieved with the two-step system.

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Abstract

L'invention concerne des procédés, des cellules, des acides nucléiques hybrides et des kits destinés à identifier ou à analyser des composés qui modifient des composants dans une cascade de signaux intracellulaires. Les procédés de l'invention consistent généralement à mettre une cellule avec un composé test. Si le composé test présente une interaction avec un composant d'une cascade de signaux pour produire une variation d'un signal intracellulaire, la variation modifie la transcription et la formation d'une protéine transactivatrice codée par un acide nucléique hybride. Le transactivateur, à son tour, se lie à un segment d'ADN parent via son domaine de liaison à l'ADN et active l'expression d'un rapporteur détectable. Ces procédés sont utiles dans le criblage destiné à l'identification de composés (p. ex. des agonistes ou des antagonistes) qui se lient à un récepteur commandant la transduction de signaux à l'intérieur d'une cellule. L'invention concerne également des acides nucléiques hybrides destinés à appliquer ces procédés et des cellules contenant ces hybrides, ainsi que des kits qui contiennent les hybrides nécessaires à l'application de ces procédés.
PCT/US2001/015426 2000-05-12 2001-05-11 Procedes et compositions destines a l'amplification de la transduction de signaux intracellulaires WO2001088194A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009000918A1 (fr) * 2007-06-27 2008-12-31 Technische Universität Dresden Dispositif et procédé de détection et d'amplification d'un signal
US20090111144A1 (en) * 2003-12-31 2009-04-30 Christopher Robert Bebbington Transactivation system for mammalian cells

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US5846722A (en) * 1996-10-16 1998-12-08 Terrapin Technologies, Inc. System to detect small molecule/peptide interaction
US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof

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US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
US5846722A (en) * 1996-10-16 1998-12-08 Terrapin Technologies, Inc. System to detect small molecule/peptide interaction

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Publication number Priority date Publication date Assignee Title
US20090111144A1 (en) * 2003-12-31 2009-04-30 Christopher Robert Bebbington Transactivation system for mammalian cells
US8354251B2 (en) * 2003-12-31 2013-01-15 Kalobios Pharmaceuticals, Inc. Transactivation system for mammalian cells
WO2009000918A1 (fr) * 2007-06-27 2008-12-31 Technische Universität Dresden Dispositif et procédé de détection et d'amplification d'un signal
DE102008030907B4 (de) * 2007-06-27 2012-07-26 Technische Universität Dresden Einrichtung und Verfahren zur Detektion und Verstärkung eines Signals
CN101743324B (zh) * 2007-06-27 2014-06-18 德累斯顿工业技术大学 用于探测和放大信号的装置和方法

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