Identification of molecules modulating protein-protein interaction

FIELD: medicine.

SUBSTANCE: group of inventions refers to methods and systems of analysis based on enzymatic degradation following protein-protein interaction for reporter modulation (activation or inactivation).

EFFECT: group of inventions provides simple high-efficiency identification of protein-protein interaction modelling.

34 cl, 17 dwg, 17 ex

The SCOPE of the INVENTION

The present invention relates to materials and methods for determining the interaction among the molecules. In particular, it concerns the ability to set, modulates whether a particular connection, e.g. "test connection", the interaction of two or more considered specific proteins. The definition involves monitoring activation of a reporter gene, which can be located in the cell, in solution or in an artificial environment or particle containing one or more of the considered reagents, where activation or its absence is called modulation or no modulation. The determination is usually carried out using the transformed or transfected cells, which also belongs to one aspect of the invention, as agents used for their transformation or transfection. You can also apply a cell-free system or a system using artificial environment or particles containing one or several analysis reagents, for example, virus, virus-like particle, a liposome, and the like.

The LEVEL of EQUIPMENT AND MATERIALS USED IN the PREPARATION of BIDS

Studies of protein-protein interactions, for example, through the identification of ligands for receptors, are great and the teres. Even if the ligand or ligands for a particular receptor is already known, interest continues to identify more efficient or more selective ligands. Receptors associated with G-protein, GPCR, also known as 7-transmembrane receptor (7TMR), will be discussed in this document as one of many examples of a class of proteins that can be characterized by the above method. However, this analysis method can be used for any proteins that interact, for example, participants metabolic pathway or cascade.

GPCR are the largest class of cell surface receptors known to man, and therefore are considered as the main application of the present invention. The ligands that modulate GPCR signals include hormones, neurotransmitters, peptides, glycoproteins, lipids, nucleotides and ions. GPCR also known as sensory receptors, such as receptors of light, odor, pheromones and taste. Due to the diverse and numerous GPCR functions are objects of intense research, for example, for chemical and biological defense and for drugs used for the treatment of various pathological conditions. Managed to achieve success in the creation of many drugs. Example is, in the work of Howard et al.,Trends Pharmacol. Sci.,22:132 140 (2001) notes that more than 50% of marketed drugs are active against these receptors.

Used in this document the abbreviation "GPCR" refers to any member of the superfamily of receptors GPCR. This superfamily is characterized by a structure containing seven transmembrane domains (7TM). Examples of such receptors, among others, include receptors class a or recopilatorio" receptor; the receptor class or Screenphone" receptors; class or metabotropic glutamatnye" receptors; receptors, belonging to the class of Frizzled and Smoothened; family of adhesion receptors or receptor EGF-7TM/LNB-7TM; adiponectin receptors and related receptors; and dynamics of receptors, including the receptors of smell, taste, Sosnica-nasal and pheromone receptors. For example, the GPCR superfamily man, among others, includes a receptor molecule, described in Vassilatis et al.,Proc. Natl. Acad. Sci. USA, 100:4903 4908 (2003); Takeda et al.,FEBS Letters,520:97 101 (2002); Fredricksson et al.,Mol. Pharmacol., 63:1256 1272 (2003); Glusman et al.,Genome Res.,11:685 702 (2001); and Zozulya et al.,Genome Biol., 2:0018.1 0018.12 (2001).

In compressed form is a common mechanism of action of GPCR function is as follows: 1) GPCR binds the ligand; 2) thereby causing a conformational change; 3) stimulates Castiglioni events which lead to changes in cell physiology. GPCR signals by modulating the activity of many intracellular proteins, for example, proteins that bind heterotrimeric guanine-nucleotide (G-protein) and β-krestinov. In the case of G-proteins, the ligand-receptor complex stimulates the exchange of guanine-nucleotide and dissociation of heterotrimeric G-protein α - and VH-subunit. In other circumstances, β-arrestin can act instead of the G-protein to counteract signaling G-protein synergistically to enhance the transmission signal G-protein etc.

For GTP-bound α-subunit and VH-heterodimer registered regulating a variety of cellular effector proteins, including adenylate cyclase and phospholipase C (PLC). In the standard cell analysis methods for GPCR receptor activity is monitored by measuring the output of the regulated G-protein-effector path, for example, accumulation of camp produced by adenylate cyclase, or the release of intracellular calcium, for example, stimulated PLC activity.

For many reasons it was difficult to develop standard methods of analysis based on G-protein signaling for some of the targets. For example, first, the different GPCR linked to different signal transduction pathways, regulated by G-proteins. Standard methods of analysis based what's on G-proteins, depend on knowledge of the specificity of G-protein to the target receptor, or such methods of analysis require genetically engineered cell systems, ensuring the participation of target receptor on effector path of the selected G-protein. Secondly, since the GPCR superfamily is so great, all cells Express many endogenous GPCR (as well as other receptors and signaling factors). Therefore, registered effector paths can be modulated by endogenous molecules, not only the target GPCR. The consequence of this phenomenon can be false positive or false negative results, for example, when trying to identify selective modulators of the target GPCR.

Regulation of the activity of a G protein is not the only result of binding of the ligand/GPCR. See, for example, Luttrell et al.,J. Cell Sci.,115:455 465 (2002) and Ferguson,Pharmacol. Rev.,53:1 24 (2001), which provides an overview of the influences that cause suppression or cessation of transmission signal GPCR. Such processes of suppression of the signal applied to prevent excessive stimulation of the cells, as well as to provide temporal relationship between extracellular signals and the corresponding intracellular path.

In General, the binding of the agonist with GPCR provides the phosphorylation of residues serine and threonine in the C-terminal part of the molecule receptor GPCR-Keene is Oh. Related in complex with the agonist phosphorylated at the C-terminal part of GPCR then interact with the family members of krestinov, for example, α-arresting, β-arresting or β-arresting-2, which inhibit or stop the transmission of the signal receptor. Binding can inhibit the interaction of the receptor with G-proteins, thereby directing the receptor for internalization and subsequent decay and regeneration. For example, linking arrestin, such as β-arrestin-2, phosphorylated GPCR may different ways to reduce the activity of the target GPCR. The simplest mechanism of inhibition by arresting its target involves binding to the intracellular domain GPCR, thereby blocking the binding site for heterotrimeric G-protein that inhibits activation of the path due to extracellular signals (desensitization). Another regulatory mechanism used by arrestingly, is the binding of the receptor with the elements of the apparatus of membrane internalization (e.g., by endocytosis-mediated clatrina)what triggers the internalization of the receptor in the vesicles with the membrane to merge with endosomes. After getting into endosome receptor may be subject to decay (e.g., lysosomes) or may be recovered in the cytoplasmic membrane, where it can once again be activated.

So you can skazat is, the binding of GPCR ligand with "modulates" the interaction between GPCR and arrestingly proteins as ligand binding to GPCR leads to the binding arrestin with GPCR, thereby modulating its activity. In this document the term "modulates" (or any derivative form) in relation to the interaction or binding simply means a change in the nature of the interaction of two proteins of the present invention, for example, in the presence of the test compound or ligand, in comparison with the nature of the interaction of these two proteins in his absence. Thus, modulation enables easy linking of two molecules. For example, the presence of the test compound may enhance or increase the degree of interaction between the two proteins, to weaken the interaction, block, inhibit, to divert, reduce or modify it in a certain way, a certain way or in a certain shape, which are amenable to detection, or the test compound can increase the probability of interaction, etc.

In some cases, the 7TMR signaling can occur without the involvement of G-proteins. Thus, when the binding of the ligand with 7TMR β-arrestin, but not G-protein is used for invoking or initiating a cascade of signal transmission in the cell. See, for example, Violin & Lefkowitz,Trends Pharm Sciences 28(8)416-422, 2007 and DeFea,Br J Pharm1-12, doi:10.1038/sj.bjp.0707508, 2007 below is a summary of two independent and dependent signal transduction pathways that begin with activated 7TMR and which can be used as a G-protein and β-arrestin; or G protein or β-arrestin.

So, for example, known antagonists 7TMR activate signal transmission β-arresting. Propranolol, a known antagonist of β₂-adrenergic receptor (ADR 2) and signal transmission G-protein turned out to be a partial agonist of the signal β-arrestin, activating transmission of a signal initiated by β-arresting, as it was established during the practical applications of the present invention.

Event signaling in the cell, reacting to external stimuli, typically mediated protein-protein interactions. Therefore, protein-protein interactions are of great interest for cell physiologists. One of the monitoring tools such interactions is an additional ribosomal protein or permuteran protein which activates a reporter, for example, the protease of the virus engraving tobacco (TEV). The split part of the reduced activity of the protease in a joint expression as fused design with interacting proteins. Wehr et al., Monitoring Regulated Protein-protein Interactions Using Split TEV",Nature Methods/i> 3:985-993 (2006). This property was used in conjunction with reporter systems related to transcription.

Understanding these mechanisms has allowed to offer alternative ways of analysis of activation and inhibition of GPCR. One such method involves the monitoring of interaction with arrestingly in the intact cell, carrying the transcription apparatus. The advantage of this approach is that there is no need for knowledge of the ways of interaction between G-protein. See, for example, U.S. patent No. 7049076: "Method for Assaying Protein-Protein Interaction", issued to Lee et al. Lee et al. reported reporter system, which needs a reporter systems associated with transcription. According to Lee et al. when the interaction of two proteins from the first protein is cleaved peptide transcription factor. The second protein is a transcription factor that activates the reporter gene. Then, the factor performs reporter function for the expense of transportation to the nucleus to carry out transcription detected by the reporter. Because the method depends on the transcription, it cannot be used, for example, in platelets, artificial environment or particles, for example, liposomes, spiral aggregates, virus-like particles and particles of viruses.

In the work of Oakley et al.,Assay Drug Dev. Technol., 1:21 30 (2002) and U.S. patent No. 5891646 and 6110693, "Methods Of Assaying Receptor Activity and Cnstructs Useful in Such Methods", issued by Barak et al., describes the methods of analysis, by which is measured the redistribution of fluorescently-labeled molecules arrestin in the cytoplasm on activated receptors on the cell surface. These methods rely on the visualization of cells with high resolution to measure relocalization arrestin and activation of the receptors. Qualified professionals will be obvious that this is a complex and time-consuming procedure, which can interfere with the affinity and the impact of complementary fragments of the enzyme used in the present invention, which may compete with the target interaction, induced by the modulator. Therefore, the disadvantage of this method is obtaining false-positive results arising from their own reassociate enzyme, regardless of the binding ligand. It would be desirable to develop a simpler, more stable method of analysis with a lower frequency of false-positive results, which could be easily adapted for high throughput screening.

Issued and sent many other U.S. patents and patent applications relating to the above-mentioned problems. For example, U.S. patent No. 6528271, "Inhibition Of β-Arrestin Mediated Effects Prolongs and Potentiates Opioid Receptor-Mediated Analgesia", issued by Bohn et al., describes analysis methods for screening Bo is autosomic funds in which measure the inhibition of binding of β-arrestin. In the published patent applications U.S., for example, 2004/0002119, 2003/0157553 and 2003/0143626; and in U.S. patent No. 6884870 proposed various forms of analysis using the GPCR. In U.S. patent No. 7128915 described similar technology GPCR. In the above U.S. patent No. 7049076, which in General concerns of GPCR functions or methods of screening, demonstrated the importance of GPCR research.

Thus, for solving problems in the field rather one of the features of the present invention, namely, providing a simpler method of analysis to track and / or determine the specific modulation of protein-protein interactions, for example, mediated by receptors of the physiological processes, such as mediated GPCR cell response, where to proteins, among others, are integrated into the membrane proteins, including receptors in General, and GPCR as an important example.

BRIEF description of the INVENTION

The present invention provides methods for determining modulates whether the test compound under consideration of specific protein-protein interactions. Protein-protein interactions is a fundamental mechanism of biology, through which the cell can interact with their environment, wnek otocnym event for example, the binding of ligand to the receptor, and can produce internal response in terms of the internalization of the ligand or without it. Internationalisation can be used two or more protein, part of which are located on the membrane or outside. Therefore, the dimer formation, heterodimer or multimer can produce internal response. Intracellular protein-protein interactions may also be involved in signaling cascades. The General scheme of the present invention is applicable to protein-protein interactions of any kind. For example, the interaction may be between two integrated membrane proteins between integrated into the membrane protein and the cytoplasmic protein, between cytoplasmic proteins, etc. In one example implementation is considered cytoplasmic protein that undergoes translocation to another organelle, for example, the nucleus where it activates a reporter for the occurrence of the signal. Preferably use analysis on a cellular basis, but may be a cell-free system, for example, involving lysates, membrane fractions, nuclear fractions, etc. in addition, there are systems with artificial environment or particles containing one or several analysis reagents, for example, liposomes, virusopodobnyh the e particles, etc. The present invention is an improvement discussed above patent Lee et al., since in this case there is no need for transcription. Therefore, the results can be obtained faster, and you can get them in cellular and cell-free systems. Below is a General description of certain particularly preferred embodiments of the invention. Examples of implementation are illustrative and in no way limit the scope of the invention as described herein and in the claims.

One of the features of the present invention, includes a contact of at least one test compound with the cell's surface, which expresses the considered protein. The test compound can be measured by its ability to modulate the activity of the studied protein, for example, the receptor protein. The expression of the protein may be the result of the transformation or transfection of the selected cells, for example, cell lines insects or mammals conducted by: (1) the nucleic acid molecule or molecules, comprising (consisting) of (a) polynucleotide, which encodes the first examined the protein, and (b) polynucleotide, which encodes an activating reporter protein containing the site cleft is to be placed, sensitive to protease, or active or activatable portion of the protease, and (2) the nucleic acid molecule or molecules, comprising (consisting) of (a) polynucleotide that encodes the second protein whose interaction with the first of the considered protein is altered in the presence of the modulator, for example, a positive current of the test compound, and (b) polynucleotide, which encodes a protease or an active or activatable portion of the protease specific cleavage site encoded by nucleic acid (1). Molecules, for example, a positive current test compounds that modulate the considered protein-protein interaction between the two proteins considered), can be evaluated or analyzed by adding, for example, optionally, the substrates activates the reporter protein in the cells expressing the first and second proteins considered, and reporter system in accordance with the description of the present invention.

Therefore, the method proposed in the present invention, can be use paratroopa enzyme for registration of the considered protein-protein interactions. Permaturely activating protein, e.g. enzyme, used as a reporter or activating the reporter protein may be in inaktivirovannoj comprising the AI, which can be activated, for example, cleavage by, for example, enzymatic activity associated with the second of the considered protein. In another embodiment uses an inactivated activating the reporter protein, which is activated by interaction of the first and second proteins considered. Thus, it is possible to carry out the screening of compounds that modulate the interaction of the first and second proteins considered. One of the advantages of this system is that it provides high-performance identification of molecules that selectively modulate specific protein-protein interactions.

The enzyme, which (alone or with one or more related molecules) to produce registered "protein a", is present in the form of activity, which can be changed. Due to this change, you can activate or inactivate the enzyme. For example, the enzyme can embed cleavage site, so as to inactivate it by splitting, for example, by a second enzyme that is associated with the second of the considered protein.

Alternatively, the splitting can lead to activation. The selected enzyme (or enzymes) can be embedded into the desired cell host through the use of one or more nucleic acids. In the example, the vector may include polynucleotide that encodes the selected molecule as inactivated enzyme, which can be activated by cleavage of the inactive enzyme at the site of cleavage. The cleavage site may have a natural origin, but preferably, it is inserted into polynucleotide that its expression led to parachironomus the enzyme. For example, in the cell-the owner can transactivate cleavage site that is not native to the protein of this cell, and (or) protein, which is not native to the host cell. Alternative embodiments include an enzyme that is activated by cleavage or by removal of the blocking peptide, either by stimulation changes the configuration of the two polypeptides for their rearrangement with subsequent activation of the enzymatic activity.

Thus, one example of implementation refers to the active polypeptide, for example the enzyme. The enzyme can be inactivated by cleavage. For purposes of specificity, it may be necessary to form the enzyme cleavage site recognized by a protease that is not native to the host cell. The cleavage site can be introduced in the form of a linker that binds, that is, keeps in contact, the two parts or two motif Fe is ment", such a linker may be the site of cleavage, native for the "enzyme", for example, the enzyme to the site of cleavage can occur from other types of cells or other organisms and absent in the cell-the owner, or the site of cleavage may be due to the conservative substitution of one or several amino acids. Conservative substitutions are well known to experts in the field. For example, charge, size, aromaticity, or other characteristics may remain unchanged to preserve activity. The activity need not be the same as the one for aperativno "enzyme", but must change at the cleavage site cleavage. The cleavage site may be inserted between the two parts of the enzyme. The cleavage site can destroy the enzyme, causing, thus, inactivation, or may allow the preservation of catalytic activity, for example, by removing the peptide portion that blocks the binding or catalytic site, or allowing two parts permuteran enzyme to interact in a manner that ensures the recovery of activity.

Therefore, the cleavage site of cleavage can inactivate or activate logged protein. The cleavage can be carried out in the presence of test compounds, for example, if the product expre what these nucleic acid molecules, which includes polynucleotide encoding the second of the considered protein, interacts with the first of the considered protein, thereby initiating the activity of the protease that recognizes and cleaves the sequence that is sensitive to cleavage by the protease in permuteran protein which activates a reporter.

The second examined the protein interacts with the first of the considered protein in the presence of a third molecule or, alternatively, in her absence. Thus, it is believed that such third molecule modulates protein-protein interactions between polypeptides a and B. Therefore, protein-protein interaction or peptide-peptide (for the purposes of this discussion, the terms "protein" and "peptide" are equivalent), which is modulated by a third molecule, for example, tested the connection, effectively detected by the system of the present invention. Molecules that modulate protein-protein interactions (between polypeptides, denoted 1 and 2, first and second, and In which expressions and definitions in this document are equivalent), you can register on active molecule that activates the reporter, or adding substrate active protein which activates a reporter, cells expressing the system, including the considered proteins.

The selection of proteins and is determined depending on the objectives, as there may be used a pair of molecules for which it is known or suspected the possibility of the Association, interaction, etc. As discussed herein, the suitable pair is 7TMR with either G-protein, or β-arresting. Another example is the Frizzled receptor and binding protein Dishevelled, etc. Another example is a protein acting during and after the interaction of cells. Means proteins a and b are located in the cell 1. If cell 1 comes into contact with the cell 2, this interaction causes the reaction cell 1 and cell 1, as indicated by the Association, interaction and other proteins a and b that further show the reagents of the present invention that produce clear and the detected signal.

Another example is the case when the protein And is expressed on the cell 1 and the protein is expressed on the cell 2. This can be achieved, for example, by means of genetic engineering, so that G protein or β-arrestin had the extracellular domain, or to activate the reporter protein had the extracellular domain, which could be activated, for example, cell 1 after cell activation 1 ligand or potential drug. Alternatively, possible spontaneous Association of endogenous molecules of the two cells. In another embodiment, the protease activating the reporter molecule p is duritsa thus, in order to be expressed on the surface of cells or particles in the form of the extracellular domain.

In yet another example implementation to proteins that are associated, aggregated, etc., with the formation of composite structures are proteins a and B. this method of analysis can be used to identify molecules that contribute to the Association or aggregation or inhibits them. An example would be the formation of the capsid of the virus, virus-like particle Assembly or the formation of ribosomes.

In the framework of the General arrangements for receptors associated with G protein-coupled (GPCR also known as 7TMR, what are the equivalent terms used in this document), the activation of GPCR agonist leads to the involvement of intracellular molecules, which are involved in the transmission signal, for example, in the initiation, termination, synergistic strengthening, resistance, etc., for example, in the case of a G-protein or β-arrestin. Therefore kinase receptor associated G-protein may act on the activated receptor, leading to its phosphorylation. Phosphorylated receptor contributes to the binding of β-arresting with the receptor. This mechanism persists for some GPCR. In other cases, the activated receptor instead reacts with β-arresting.

To assess the reactivity of molecules, Modulare the observed protein-protein interactions, for example, activation of the GPCR, has developed a system of analysis of protein-protein interactions, which was tested on the system GPCR-permuteran reporter molecule. For example, a reporter molecule can be a system for analysis of luciferase/luciferin. Generally, as the reporter molecule is exogenous molecule that is foreign to the host cell or the mechanism of signal transmission. Due to this it is possible to minimize the spontaneous activation of the reporter molecule the host-cell and cell-host and, thus, the generation of the signal, and thus false positive results. Activating the reporter molecule can have a domain structure or may permutates to produce an inactivated protein which activates a reporter who can show reporter activity under certain manipulations. Therefore, in the present application refers to the use of latent molecules that activate the reporter. Permuteran molecule that activates the reporter, minimizes spontaneous activity of a molecule that activates a reporter, and therefore false positive results. For example, when analyzing complementaly fragments of the enzyme affinity fragments of the enzyme can dominate the kinetics of the reaction with the molecule-target ligand or molecule that passes the TFR is ning, so what happens spontaneous reassociate fragments of the enzyme in a functional molecule that contributes to a higher background and (or) false-positive results. Consider permuteran protein which activates a reporter, can be designed to contain the site, which is under the influence allows permuteran molecule that activates the reporter, to form a functional molecule. This site may be the site of the protease. The site of the protease is preferably a unique website, which is rarely present or absent in the cell-the owner or particle, which is a component or components of this method. Due to this, provide an additional means to avoid spontaneous reassociation intact molecules that activate the reporter, and thus minimized false positive results. The specific signal is observed only when the ligands, ultimately, induce protease in the immediate vicinity of the inactivated protein which activates a reporter for splitting it, and only in this case can be formed object, activating or generating an active signal. Experts in the field, the number of known proteases that can be used for practical applications of the present invented who I am. For example, can be used protease from viral sources, because they, as a rule, are alien to the intact host cell. In one application is used gene permuteran protein which activates a reporter, in which the coding sequence of the Firefly luciferase coupled with the C-terminal end of the sequence GPCR, and β-arrestin-2 (Ar2 or Arr2) associated with the gene of the protease of the virus engraving tobacco (TEV). In another example implementation permuteran luciferase coupled with β-arresting (Ar or Arr), and gene TEV protein bound on the transmission path of the signal, reacting with β-arresting, or receptor, for example, 7TMR, presumably acting independently from G-proteins. If a plasmid is designed to Express both of the above protein is subjected to expression in cells, compounds modulating the interaction of GPCR-arrestin-2, attract Arr2 protein-protease site in the protease recognition in permuteran the luciferase, and the TEV protease cleaves permuteran the luciferase. The effect of the test compounds can be measured by the change in enzymatic activity, which is achieved through the reconstruction of a protein which activates a reporter, in this case, the luciferase becomes active and can generate a detected signal due to the impact of the approach is a first substrate, for example luciferin.

The scope of the invention is not limited to luciferase, or even enzymes. Activation through cleavage refers to well-known phenomena, such as proenzyme. You can also use non-enzymatic reporter systems. For example, can be used green fluorescent protein (GFP). Permuteran GFP, for example GFP with moving parts, can play the role of a protein which activates a reporter, and reporter. The action of the protease, for example, TEV or other protease with its website recognition, included in permuteran peptide, leads to the splitting of permuteran protein which activates a reporter/reporter, thereby making it possible reorganization, which produces the signal. GFP itself has the advantages of a detectable molecule that transmits a signal to the reporter. Alternatively, in the reporter molecules can enter the sites of cleavage, which do not exert any appreciable disturbance to the signal. Splitting resulting from protein-protein interaction then leads to a decrease of the signal of the reporter. In the design of the reporter may be multiple sites of cleavage.

The tertiary structure of the protein can be useful for a qualified expert in the area when determining the place of introduction of the cleavage sites. For example, if two parts is of polypeptide are in close contact, it can be expected that the separation of these parts due to the violation of the sequence will reduce or eliminate the activity. It can be assumed that after the splitting of these parts will interact that will lead to recovery.

First consider the protein may be integrated into the membrane protein, for example, transmembrane receptor-like GPCR. Examples of transmembrane receptors include β-adrenergic receptor (ADR 2), the receptor for arginine-vasopressin-2 (AVPR2 or V2), the receptor of serotonin-1a (HTR1 A), muscarinic acetylcholine receptor m2 (CHRM2), a receptor of the chemokine-5 (C-C motif) (CCR5), the receptor dopamine D2 (DRD2), Kappa-opioid receptor (OPRK) or α1a-adrenergic receptor (ADRA1A), etc. are Integrated into the membrane receptors are well known to experts in the field. It should be understood that in all cases the scope of the invention is not limited to the specific embodiments described in the form of examples of the present invention. For example, such molecules as the receptor for insulin-like growth factor-1 (IGF-1R), which represents tyrosinekinase, and proteins, which in the normal state are not integrated into the membrane, like the estrogen receptor-1 (ESR1) and estrogen receptor-2 (ESR2), can also be used in the present invention. Protease or a portion of a protease associated with the protein, can the t to be a protease nuclear inclusion virus engraving tobacco A (TEV). TEV has a website recognition, containing seven amino acid residues, and therefore more specific than protease with smaller, statistically more common sites recognition. Other proteases are also suitable for use in the present invention. For example, enterokinase and protease factor Xa, each of which has the sequence recognition with five amino acid residues, thrombin and PureAct^TMor Clean Cut^TM, each of which has the sequence recognition of the six amino acid residues, and PreScission^TMsequence recognition of the seven amino acid residues are proteases that can be used in the present invention. The present invention is not limited to use of any specific protease. Thus, the protease must provide a breakdown on the website, which leads to the generation or change of the signal from the reporter.

As a protein which activates a reporter can be any enzyme that can act on the substrate to generate a detected signal. For example, the enzyme may directly or indirectly increase or decrease the fluorescence or chemiluminescence, or may cause discoloration. Reporter substrate can be of a biological nature, for example, a protein, or it may be him who ical connection the reaction which catalyzes the reporter enzyme. The second examined the protein may be inhibitory protein, such as arrestin. Arrestin usually interact with the GPCR to modulate activity in response to the interaction of the ligand with the receptor. The cell can be a eukaryotic or prokaryotic. As a reporter can be exogenous component, for example, β-galactosidase or luciferase. For simplicity, the term "reporter enzyme" is used as equivalent molecules that activate the reporter activator reporter; molecules for modulation of the reporter protein, modulating the reporter; or a protein which activates a reporter, as well as the abbreviated name of the molecule, which leads to changes in the output of the reporter. For example, the reporter enzyme can permutative to cause a change in reporter signal or, for example, can fermentative or afermative to cause a signal change, for example, the fluorescence signal. A qualified specialist in the field have an understanding of various reporter systems and proteins that modulate or activate the reporter signal.

The nucleotide sequence encoding a first protein may be modified in order to increase the interaction with a second protein. Such modifications, among others, include full or hour the ranks replacing the nucleotide sequence of the C-terminal region of the first protein nucleotide sequence, which encodes the amino acid sequence that has a higher affinity for the second protein, compared with the original sequence. For example, the C-terminal region may be replaced by a nucleotide sequence that encodes a C-terminal region AVPR2, AGTRLI, F2RL1, CXCR2/IL-8b or CCR4. Such modifications are known to experts in the field and determine additional characteristics of the present invention.

The methods of the present invention may include the interaction of multiple test compounds with many samples of cells or particles. Each sample can interact with one or more test compounds. In another embodiment, the cell or particle contain two different molecules with extracellular domains, including a variety of molecules that activate the reporter, and they both interact with β-arresting. The screening is conducted by determining activity of a reporter, for example, by monitoring the enzymatic activity in samples in order to establish modulate whether any compound or mixture of compounds of specific protein-protein interactions. The method may include the interaction of each test sample with the same test compound, the interaction of each test sample with the mixture of test compounds or can combine these approaches. By using the methods of the present invention can conduct tests or screening for compounds that inhibit the binding of substances with protein A. for Example, the analysis may include a known ligand protein a, and can identify and / or characterize compounds that modulate the binding of ligand to the protein, as well as in the method of competitive analysis. Control samples can be used in each test procedure, or can be analyzed in parallel.

In some embodiments of the present invention provides methods for determining modulates whether the test compound is one or more kinds of the considered protein-protein interactions. For such embodiments, as a rule, is characterized by the interaction of test compounds with a variety of sample cells that have been transformed or transfection: (a) the first nucleic acid molecule, including (i) polynucleotide, which encodes a first protein and a polynucleotide sequence encoding the cleavage site of the protease, and (ii) polynucleotide, which encodes a protein which activates a reporter in the cell; and (b) a second nucleic acid molecule, including (i) polynucleotides, encoding the second protein whose interaction with the first Bel the om in the presence of the considered the test compounds to be measured, and (ii) polynucleotide that encodes the protease or a polypeptide that is specific for the cleavage of the polypeptide at the site of cleavage. In many samples the first protein may differ from other first protein. Then the method includes determining the activity of the reporter in one or more of a variety of patterns by determining the modulation of one or more interactions of the considered proteins.

The second protein may differ in each sample, or to be the same in all samples. All samples can be combined in a common receiver, and each sample can have a different pair of the first and second proteins. Alternatively, each sample may be tested in different receiver. Reporter in a particular sample may differ from the reporter in the other samples. The mixture of test compounds may include a biological sample or present in them, for example, cerebrospinal fluid, urine, blood, serum, pus, ascitic fluid, synovial fluid, tissue extract, plant or herbal extract or exudate.

In other embodiments of the present invention features a recombinant cell transformed or transfectional (a) a nucleic acid molecule comprising (i) polynucleotide, which encodes a first protein, (ii) polynuclear is d, encoding the cleavage site of the protease, part of the protease or a polypeptide with by activity, and (iii) polynucleotide, which encodes a protein which activates a reporter in the cell; and (b) a nucleic acid molecule comprising (i) polynucleotide that encodes the second protein interacting with the first protein in the presence of the test compound to be measured, and (ii) polynucleotide, which encodes a protease, a portion of the protease or a polypeptide with by activity specific to a given site of cleavage.

One or both of the nucleic acid molecule can be stably integrate into the genome of the tested host cell. The cell can be transformed or transactivate with the reporter. The first protein may be integrated into the membrane protein, for example, transmembrane receptor-like GPCR. Examples of transmembrane receptors include ADR 2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, OPRK or ADRA1A.

As noted above, in the role of protease or portion of a protease may be not only protease nuclear inclusion virus engraving tobacco, but any protein that activates protein which activates a reporter, and can be any enzyme that acts on the substrate in order to obtain an acceptable or detected signal. The second protein may be inhibitory protein. The cell can be a eukaryotic or procario the Ohm, usually for screening pharmaceuticals preferable to use eukaryotic cells. Particularly preferred cells that glycosylase by a mechanism similar to the final target of a pharmaceutical product. Cells can be grown or be designed in such a way as to achieve the desired glycosylation. The use of prokaryotic cells that do not have characteristics of glycosylation relevant offer the ultimate target may be useful for screening and characterization.

As a reporter can be exogenous component, for example, β-galactosidase, GFP or luciferase. The nucleotide sequence encoding a first protein can be modified so as to increase the interaction with a second protein, for example, for this purpose you can make full or partial replacement of the nucleotide sequence of the C-terminal region of the first protein nucleotide sequence that encodes the amino acid sequence that has a higher affinity for the second protein, compared with the original sequence. The C-terminal region can be replaced with a nucleotide sequence that encodes a C-terminal region AVPR2, AGTRLI, F2RL1, CXCR2/IL-8B, CCR4 or GRPR.

Within the more real the embodiment of the present invention features an isolated nucleic acid molecule, includes (i) polynucleotide, which encodes a protein, (ii) polynucleotide encoding a cleavage site for a protease, part of the protease or a polypeptide with by activity, and (iii) polynucleotide, which encodes a protein which activates a reporter in the cell or in another system for analysis. As protein can be used integrated in the membrane protein, for example, transmembrane receptor-like GPCR. Examples of transmembrane receptors include ADR 2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, OPRK or ADRA1A. Protease or a portion of the protease may be a protease nuclear inclusion virus engraving tobacco A. As noted above, in the role of a protein which activates a reporter can be any protein that interacts with the substrate to generate a signal, and it is not necessarily limited to the example TEV discussed in this document. This or another example of the present invention should not be construed as limiting the scope of the invention to a specific set of examples of implementation.

In some embodiments of the present invention features an expression vector containing an isolated nucleic acid molecule that includes (i) polynucleotide, which encodes a protein, (ii) polynucleotide encoding a cleavage site for a protease, part of the protease or a polypeptide with by activity, and (iii) poly is ucleotide, which encodes a protein which activates a reporter in the cell and functionally linked to the promoter.

In some embodiments of the present invention features an isolated nucleic acid molecule that includes (i) polynucleotide that encodes a protein whose interaction with another protein in the presence of test compounds is supposed to measure, and (ii) polynucleotide encoding the protease, part of the protease or a polypeptide with by activity, it is specific to the site of cleavage. In the role of the studied protein or other protein can be inhibitory protein, such as arrestin.

In some embodiments the present invention also includes an expression vector containing an isolated nucleic acid molecule that includes (i) polynucleotide that encodes a protein whose interaction with another protein in the presence of test compounds is supposed to measure, and (ii) polynucleotide that encodes the protease or a portion of a protease which is specific to the site of cleavage; and the specified nucleic acid, in turn, functionally linked to a promoter.

Another exemplary embodiment relates to fused protein obtained by expression: an isolated nucleic acid molecule comprising (i) polynucleotide, the cat is which encodes a protein, (ii) polynucleotide encoding the cleavage site of the protease, part of the protease or a polypeptide with by activity, and (iii) polynucleotide, which encodes a protein which activates a reporter in the cell, and which is functionally linked to the promoter; or an isolated nucleic acid molecule comprising (i) polynucleotide that encodes a protein whose interaction with another protein in the presence of the test compound to be measured, and (ii) polynucleotide, which encodes a protease or a portion of a protease that is specific to the site of cleavage.

In other embodiments of the invention it is proposed to use a set of tests used to establish modulates whether the test connection of the considered protein-protein interactions. In the set of tests includes one or more of the following components: a separately packaged quantity (a) nucleic acid molecules, including polynucleotide, which encodes a first protein (i) polynucleotide encoding the cleavage site of the protease, part of the protease or a polypeptide with by activity, (ii) polynucleotide, which encodes a protein that activates reporter gene in the cell; and (b) nucleic acid molecule comprising (i) polynucleotide that encodes the second protein, the interaction of which with a first protein in the presence of ispytav is imago compounds to be measured, (ii) polynucleotide, which encodes a protease or a portion of a protease that is specific to the site of cleavage; which is set as a possible option provides for the storage of all items (a) and (b) separately from each other. The set can be accompanied by instructions for use. Alternatively, the kit may contain cells engineered to Express either or both of the considered fused protein.

The first protein may be integrated into the membrane protein, for example, transmembrane receptor. Specific transmembrane receptor is a GPCR. Specific transmembrane protein is a GPCR. Examples of transmembrane receptors include ADR 2, AVPR2, HTR1A, CHRM2, CCR5, DRD2, OPRK or ADRA1A. Protease or a portion of a protease or a polypeptide with the activity of the protease can be a protease nuclear inclusion virus engraving tobacco A. Protein that activates the specified reporter can be, for example, a protease that acts on detektiruya molecule that activates the reporter, responsive to activation by cleavage. The reporter can be any molecule that produces the detected signal of the cleavage product. The second protein may be inhibitory protein, such as arrestin. The kit may also include separately packaged quantity isolated forefront of the crystals of nucleic acids, which encodes a gene that activates the reporter. As activator of the reporter may be, for example, β-galactosidase or luciferase. The nucleotide sequence encoding said first protein can be modified so as to increase the interaction with a second protein, for example, for this purpose you can make full or partial replacement of the nucleotide sequence of the C-terminal region of the specified first protein nucleotide sequence that encodes the amino acid sequence that has a higher affinity for the second protein, compared with the original sequence. The nucleotide sequence of the indicated C-terminal region can be replaced with a nucleotide sequence that encodes a C-terminal region AVPR2, AGTRLI, F2RL1, CXCR2/IL-8B and CCR4.

Assume that any method or composition, described herein, can be used for any other method or composition described herein. The use of the singular with the definition of "containing" in the formula and (or) the description of the invention may relate to a single number, but also in meaning does not contradict the definitions of "one or more", "at least one" and "one or more". Where appropriate components are used is of slightly different wording, they do not necessarily identify different examples of implementation, but taken together, describe the corresponding elements in the broad sense.

These and other embodiments of the present invention will be more and more deeply understood from the subsequent description and the accompanying drawings. However, it should be understood that the following description, though, and refers to various embodiments of the present invention and many of its specific details are given only for illustration and not restrictive in character. In the description of the invention may be made of various substitutions, modifications, additions and / or rearrangements that do not affect its essence, and the present invention covers all such substitutions, modifications, additions and / or changes.

BRIEF DESCRIPTION of FIGURES

The accompanying figures are part of the present description and are incorporated in order to further demonstrate certain aspects of the present invention. Consideration of one or more given shapes in combination with the detailed description of specific embodiments presented herein can facilitate understanding of the present invention.

In Fig. 1 provides a schematic description of one of the embodiments, in which the modulator4of binding the t-associated protease protein 1that makes this protein to interact with a second protein2associated with reporter modulator3for example, permuteran inaktivirovannye modulating protein. Modulator4is the compound that modulates protein-protein interactions. In the present example, for example, Ar or arrestin2merged with inaktivirovannye permuteran protein3that modulates or activates the reporter. Protease7associated with protein1. Shows the site of cleavage8between the two segments of the protein that modulates reporter3.The cleavage of the protease after interaction with the modulator4associated with protein1for example 7TMR, the activity of a protein which activates a reporter, restored, which, ultimately, leads to detectivemisa signal, which is indicated by the light6.

In Fig. 2 is a diagrammatic representation of the analysis of protein-protein interactions of the present invention. Integrated into the membrane protein21related intracellular protease22communicates with the modulator4. An inactivated protein23associated with the reporter carries an inactivated reporter3or activator reporter3. When interacting protease22associated with protein21splits the site of cleavage permuteran protein23, enable the subsequent reporter, that provides for the reorganization of the5parts of the protein molecule activates a reporter protein3after the modulator4binds protein21. Reporter activator provides the reconstruction of the reporter or activator reporter3to invoke or activate reporter signal.

In Fig. 3 schematically shows an exemplary embodiment in which the two interact transmembrane protein. Molecule4causes (modulates) the interaction between two membrane proteins, at least one of which, for example, is a receptor protein. This figure protease22for example, TEV (7in Fig. 1), associated with protein1and is in close proximity to permuteran fused protein3activating the reporter and associated with a second membrane protein33. Proteolysis at the cleavage site 8, which is provided by the proximity of proteins1,33leads to activation of the5protein3activating the reporter.

In Fig. 3 also shows a schematic application of the technology identification of molecules, modulating the formation of glycosilated or heterodimer receptor. Shown in the diagram proteins1and33refer to the integrated membrane proteins. Proteins1and33were designed in such a way that each of them included either protease2 or the reporter3activated protease22. Molecule4modulation of the interaction that binds, for example, protein1and (or)33. When interaction1and33protease22affects the activator reporter3thereby leading to changes in the signal. Heterodimerization can distribute coverage of traditional homodimerization. For example,1and33can represent two copies of the same receptor, but differ by binding to protease or a reporter. As already mentioned, the definition of "reporter" and "activator reporter" are often used interchangeably to describe various embodiments of the present invention.

In Fig. 4 is an example, including protein-protein interactions for intracellular proteins. Protein41is, for example, a nuclear hormone receptor, fused to the activator22for example TEV. Permaturely molecule43activating the reporter, localized in the cell nucleus40. The reporter can be localized in the nucleus at the expense of the basic polypeptide that acts as a peptide sequence for nuclear localization. After binding of the ligand, for example, a hormone (not shown), there is a translocation merged with other41in the nucleus, where it interacts with the reporter system.

In Fig. 5 shows that the activity regen the new luciferase, defined permuteran fused luciferase proteins by TEV protease in the cells can be controlled by modification of the cleavage site of the protease. Thereby, it becomes possible to adjust the signal-to-noise. The two designs, ADR 2 is a β2-adrenergic receptor, Luc 234-550 and 2-233 are two fragments of luciferase related through X, site of TEV cleavage with varying C-terminal amino acid. As X may be, for example, serine, S, arginine, R or valine, V. reconstructed Activity of luciferase from permuteran fused protein luciferase was observed in mammalian cells, if a cell has attended both designs. Exposure to decomposition when exposed TEV may depend on specific amino acid residues at the cleavage site. Here, and in other cases, RLU denotes particles with relative luminescence (or light). In this experiment has not been used for the ligands.

In Fig. 6 shows the dependence of agonist-induced luciferase activity, according to the cellular analysis of GPCR-permuteran luciferase using ADBR2 receptor associated with permuteran luciferase with different cleavage sites of the protease TEV, R at position X site cleavage TEV (left) and V at position X (right graphy is). The TEV protease was merged with arresting. On the x-axis on each graph deferred to zero (no agonist) and in the presence of 10 μm agonist.

In Fig. 7 is dose-dependent response of luciferase activity, according to the cellular analysis of GPCR-permuteran luciferase in systems with joint intermediate transfection and partial intermediate transfection. In the left graph in cells SNO design was used with valine at the cleavage site of the protease. Conducted joint interim transfection of constructs ADR 2-luc and Arr-TEV. On the right graph cells stably was transfectional R-containing structure luciferase, merged with ADR 2, and then spent an intermediate transfection design Arr-TEV.

In Fig. 8 provides an alternative method of analysis of GPCR-permuteran luciferase. Construction of expression contained ADR 2 associated with the activator reporter, and arrestin-2, associated with the protease. Construction of the intermediate was transfectional in HEK 293 cells. Reaction kinetics displayed on the graph in the case of one hour (▲) and five hours (■) incubating the reaction.

In Fig. 9 depicts the process of generating line of HEK cells stably expressing the design arrestin/permuteran enzyme containing V is at site X, which were intermediate transfection receptor ADR 2-TEV (left)and line of CHO cells stably expressing Arr-luc234S233 and intermediate transfection design of receptor-TEV.

In Fig. 10 provides an assessment of the analysis Per-Luc for the agonist (isoproterenol) (■), partial agonist (▲), antagonist plus isoproterenol (▼), antagonist (♦) and non-specific endogenous receptor (●): responses in HEK cells stably expressing the design arrestin/permuteran enzyme and intermediate transfection ADR 2-TEV.

In Fig. 11 shows the estimation results of the analysis of GPCR Per-Luc with agonist V2 (vasopressin receptor-2) and the inverse agonist. The HEK cells stably transfection design arrestin/permuteran luciferase, were intermediate transfection design V2-TEV and are induced by the agonist, 8AVP, arginine-vasopressin (left). When these cells were tested in the presence of the inverse agonist, was observed dependence on dose, and the signal was mediated by arresting, but not G-protein (right panel).

Fig. 12 is devoted to several analyses ADR 2 on the basis of β-arrestin. The graph to the right cells DiscoveRX HEK were consistently transfection ADR 2 in accordance with the manufacturer's instructions and tested antagonist (propranolol) (■), agonist (isoproterenol) (▲) and their combination (●) on the left and right agonist (■), inverse agonist (▲), right) and their combination (●). The results of the analysis to the right, were compared with the results of the analysis of the present invention to reply n the antagonist, the agonist isoproterenol and their combination (left). The considered analysis on the left provided a higher level of discrimination and higher specific activity.

Fig. 13 reflects the analysis of V2 on the basis of β-arrestin. The graph of the inverse agonist V2 (SR121463) induces a signal-independent G-protein, but dependent on arrestin.

In Fig. 14 shows the expression of constructs that contain overlapping essentially the full length, but not exact copies of luciferase, United, as proposed herein, to get permuteran the luciferase. CMV denotes the promoter of cytomegalovirus. Luc2-456 and Luc234-550 are essentially full-length fragments of luciferase. In this example,

GS corresponds to the peptide linker consisting of glycine and serine. On the site of TEV cleavage at the C-terminal end is valine.

In Fig. 15 provides an analysis for monitoring intracellular protein-protein interactions. Rapamycin is an immunosuppressant, which simultaneously binds to rapamycin-binding protein (FKBP12 or FKBP) and FKBP-rapamycin-binding (FRB) domain of the target of rapamycin kinase mammals (mTOR). mTOR is a mouse serine/ser / thr protein kinase, containing rapamycin-binding domain151that is the target of rapamycin ml is capitalship 154. FKBP152means FK506-binding protein with a weight of 12 kDa, which contains the binding site of rapamycin. TEV-protease merges with the rapamycin-binding domain of mTOR, FRB151. Permuteran protein which activates a reporter, merges with FKBP152the rapamycin-FKBP12 binding domain. Rapamycin154reacts with FRB151and FKBP152that mediates binding to them and delivers them in the immediate environment, which leads to activation permuteran reporter.

In Fig. 16 is a diagram of the analysis, where the proteins A21and B23are integrated into the membrane of the receptor, which spontaneously dimerized (left to right) or dissociate (right to left) when the binding of the ligand. Analysis allows you to monitor the spontaneous interaction of two receptors or induced interaction, in which one or both receptors bind ligand, the ligands may be the same or different. Alternatively, protein A21and B23can timeresults spontaneously or without mandatory binding ligand or modulator (not shown). In this embodiment, the protease and part permuteran activator reporter fused protein expressed on the cell surface or outer part of the artificial environment or particles. The analysis can also be carried out in about the time, to monitor the violation of receptor interaction, spontaneous or mediated by one or more molecules that can be seen on the decrease, reduction or loss of signal.

In Fig. 17 displays the data for cell analysis, where proteins are A171and B172remain inside or on the surface of individual cells, which may contact, touch, interact, etc. Again, A171or B172can transfer protease177or permuteran protein173activating signal. The analysis can be used to detect spontaneous interaction of two labeled receptors on two cells or induced interaction in which either or both receptors bind ligands that may be different or identical, indicating that the interaction or the convergence of the cells. In this embodiment, the protease177and part of the173permuteran activator reporter fused protein expressed on the cell surface or outer part of the artificial environment. Activated permuteran reporter175obtained from the protein A171and B173that are associated with each other. Alternatively, the slit receptor proteins and considered merged intracellular proteins can be in one cell, and inducing factor, ligand, etc. that is being monitored, the expression is associated to the second cell or the second cell. The analysis can also be carried out in such a way as to monitor the violation of the interaction between receptors and cells, spontaneous or mediated by one or more molecules that can be seen on the decrease, reduction or loss of signal.

DETAILED DESCRIPTION of SEVERAL EXAMPLES of IMPLEMENTATION

In the analysis of the present invention detects protein-protein interactions, and does not require prior information about the compounds modulating the interaction or cellular transmission signal, initiated by the interaction. The analysis can be used to detect the interaction of membrane proteins, for example, the formation of homodimers or heterodimers. The analysis allows to detect the interaction of a membrane protein with a cytoplasmic protein. The analysis can be used to detect the interaction of two cytoplasmic proteins. The analysis allows to detect the translocation of the protein in the intracellular compartment or organelle inside the cell. The analysis can be used to detect the interaction of two cells or environments, or particles. Each of the proteins a or b can bind the ligand, cofactor or other connection, a molecule or substance that may be important or necessary for protein-protein interactions.

The term "sequence" has several uses in genetic ingeneri is, in the field of nucleic acids and proteins that are known to experts in the field, and may have different meanings in the contexts of a sentence, paragraph, concepts, ideas, wording, etc. for Example, the sequence may be a specific list of amino acid residues of the polypeptide (primary structure) or nucleotide base of polynucleotide. In another context, the term "sequence" can refer to a complex molecule in a General sense, the example is a polypeptide sequence that applies to the entire molecule, and the knowledge of the primary amino acid structure is optional. Genetic sequence can be synonymous gene and belongs to polynucleotide as such or as a whole. Sequences can be assigned to individual polypeptides, or polynucleotides, or their fragments. Therefore, the expression "sequence functionally linked" means that certain genes, domains, or organizational units of transcription may be associated or connected in a functional way, in order to ensure the expression of a particular gene (genes), domain (domain), units (units) transcription, etc. resulting from the connection or binding. Sequences can also be a part of the specific downregulation of the gene or protein, for example, domain (Domino the protein, which has a number of functional elements or domains. As is known to experts in the field, as considered polynucleotides can be DNA or RNA, or a mixture thereof, and methods of their preparation and use in the practice of the present invention are also well known.

For example, in Fig. 4 shows analysis data modulated activity of a nuclear hormone receptor. The transport of nuclear hormone receptor to the nucleus induces or stimulates a signal through the reorganization of the protein which activates a reporter, and, in one embodiment, the molecule which is amenable to detection and can therefore act as a reporter, for example, the change or the formation of fluorescence as a result of response to the activity of activator protein, such as luciferase. The emerging signal can represent any detective change, for example, a change in intensity or change of excitation/emission. Other well-known reporter signal is chemiluminescence. A qualified specialist in the field will appreciate the fact that the protease or permuteran reporter can be designed to have an appropriate nuclear or other polypeptide-target. Such polypeptide-target may include basic amino acids. The signal to be modulated PR is the interaction of both in the target area.

In the case of GPCR present analysis method is specific, sensitive and does not require preliminary data on specific G-protein binding. The analysis is not affected by endogenous GPCR, and it can be used to identify molecules, including agonists, antagonists and inverse agonists (for some receptors). Analysis method of the present invention is an improved modification in comparison with the method of Lee et al., without the necessary gain at the expense of transcription. The present invention provides a more rapid and direct reading of the evidence.

The present invention provides a more simple and reliable system of analysis compared with those reported in Lee et al., partly because this system does not require the translocation of the reagent in the nucleus with subsequent transcription to amplify the signal. So check results can be made immediately after the act of modulating receptor. In contrast to the method of analysis of Lee et al., in the present invention is not required to engage the core. Actually, one of the applications of the present invention is the detection of secreted proteins. The reporter or protease in the cytoplasm can activate (or inactivate) the signal from the secreted protein partner. Another embodiment provides for the use without the nuclear cells or artificial cells, environment or particles.

The present invention is particularly useful for identifying molecules for modulation of any protein-protein interaction. For the method of analysis DiscoveRX^TMthat uses β-arrestin requires two interacting protein component for generating a signal all the time to stay connected. Most previous methods of analysis of GPCR relied on the signal transmission G-proteins, for example, methods of analysis FLIPR and camp. Any molecules that affect the levels of Ca⁺⁺or camp, tend to generate false-positive signals. On the other hand, proposed in the present invention, a method of analysis distinguishes speed, reliability and low cost, while it does not depend on placing the enzymatic component or signal G-proteins, which may affect the sensitivity and specificity.

The present invention provides methods of analysis or screening of any protein-protein interaction through the fusion protein A (or protein) activates the reporter protein (the terms "protein/molecule, modulating reporter", or "protein/molecule that activates reporter"are the equivalent to the above definition). An example is permuteran enzyme containing a proteolytic cleavage site. Protein is fused with FR what asoi. The interaction between protein a and protein can be constitutive or to the induction of the third molecule. A qualified specialist in the field can use the analysis method of the present invention to identify molecules that enhance or disrupt protein-protein interactions. Alternatively, protein a may combine with the protease and protein b may combine with the protein which activates a reporter.

In the present invention as activating the reporter protein protein is used, which is latent and can be activated by interaction with a second protease protein. Our approach is to obtain activating the reporter protein, which is permuteran molecule, designed to enable the cleavage site of the protease. The splitting part of the protein which activates a reporter can associate, aggregate, etc. so as to form an active polypeptide or unit that activates the reporter. It is active, for example, fermentations active protein which activates a reporter can then act on a suitable substrate, for example, the reporter in order to produce the detected signal. So, for example, if permuteran inactivated molecule is luciferase, at her cleavage with the formation of biologically and the active enzyme derived luciferase can impact on a suitable substrate, for example, luciferin, generating the detected signal, which in this case represents the luminescence.

In another embodiment, a protein which activates a reporter is a reporter. Therefore, this case can be viewed as a self-activating activating the reporter protein at the cleavage. An example can be a GFP that when splitting reorganized and enables the generation of a detected signal independent of the reporter system, for example, as in the case of the reagent, which produces luciferin, if the activator reporter is luciferase.

Permuteran genes that activate the reporter, can be designed in an active or inactivated form. For example, during the development of this technology was designed GPCR-inactivated permuteran fused with luciferase reordered amino acid sequence of the luciferase. The source of the N-terminal fragment has been moved to the C-terminal end, and the original C-terminal fragment was moved to the N-terminal end, and website recognition protease was used for merging two fragments with the modified order. The interaction of GPCR-inactivated permuteran fused protein with luciferase fused protein β-arresting-2-TEV protease leads to the splitting of inaktivera the Noah permuteran luciferase and generating the reconstructed activity of luciferase. In the frameworks of alternative strategies, the authors of the present invention introduced the design of GPCR-active permuteran fused luciferase, where the site of the protease recognition is entered in the original ordered sequence of luciferase, which does not have a significant effect on the luciferase activity. The interaction of GPCR-active permuteran fused protein with luciferase fused protein β-arresting-2-TEV protease leads to the splitting of the active permuteran luciferase and production of two fragments inactivated luciferase, which causes a loss of activity, and therefore, the reduction or disappearance of the signal.

Protein which activates a reporter can get out of peratrovich reporters protein-based, for example, luciferaseGaussia; luciferaseRenilla; β-lactamase; β-galactosidase; and fluorescent proteins, for example, one of the green fluorescent protein (GFP) or protein DsRed, etc. containing, for example, a proteolytic cleavage site, such as a site of TEV cleavage. Although the term "enzyme" is used as a General definition, activating the reporter proteins themselves are not limited to enzymes, but these are any activating the reporter proteins that are able to cause a signal change. For example, the binding or holding fluorescent white is and may result in a noticeable change in the signal without chemical reaction, changing the molecular structure.

One of the features of the present invention is the fact that qualified professionals in the field of molecular biology or protein chemistry will be able to construct variants permuteran luciferase using different break points and different areas overlap, so as to reduce or increase proteasome activity, the activity of the basal luciferase activity reconstructed luciferase. Cm. the work of Rachel B. Kapust, et al.Biochemical &Biophysical Research Communications, 294 (2002) 949-955.

Protease known to experts in the field and can come from various sources, for example, bacteria, yeast, fungi, plants, insects, mammals and other Organisms need proteases for processing of peptides, and therefore in biological systems there are many different proteases suitable for use in the present invention. The selection of the suitable cleavage sites for the desired enzyme can usually be done on the basis of data from the scientific literature or catalogues. Such cleavage sites of proteases are oligopeptides of different lengths, for example, of two amino acids, three amino acids, four, five, six, seven, eight, nine, ten or more amino acids, etc.

Permuteran protein which activates a reporter, can takesimana alternative cleavage sites of the protease or be associated with one or more inactivated pre - or proenzyme, which can be transformed into an active enzyme after cleavage. For example, the sites of cleavage of the pre - or proenzymes can be modified in order to be sensitive to the enzyme that recognizes the sequence differs from the native type. Alternatively, the cleavage site can be modified to fit specific desired effect, for example, higher specificity, higher sensitivity to splitting, etc.

The analysis can also be carried out using the active enzyme site of protease cleavage, which, after cleavage transformed into an inactivated enzyme. This feature provides a certain simplification, as many proteolytic enzymes with different levels of specificity can then affect the active enzyme as required, with minimal design changes.

Mammalian cells, e.g., HEK293, COS-7, NIH3T3, etc. and yeast cells can be used to detect protein-protein interactions in the method of analysis permuteran protein which activates a reporter. Can also be used in a cell-free system. Such cell-free systems include lysates, membrane preparations, viral material, virus-like particles, liposomes, platelet drugs is s membranes, spiral units, other artificial particles lipid based or environment, which modulate the biological membrane with the formation of patterns that can capture, link, move, enable, etc. a particular biological object, such as a transmembrane protein. In this method of analysis can be used by living organisms, for example, transgenic organisms, or they may be selected cells or reagents that can be used in the present method of analysis.

In this method of analysis also serves detected reporter. This reporter is a substrate for this protein which activates a reporter. Therefore, in the case permuteran the appropriate luciferase reporter is an enzyme, which when exposed to a luciferase produces the detected luminescence signal. The reporter can be intracellular to provide a method of analysis that allows to do without cell lysis. For example, GFP, fused with the carboxy terminal residue maltose binding protein (MBP) is not fluorescent in the presence of the signal sequence of the MBP. When removing the signal peptide MBP observed fluorescence. Cm. work Feilmeier et al., J Bacteriol 182(14)4068-4076, 2000. Therefore, after the signal peptide MBP, you can enter the site of cleavage of the protease is, as proposed in this document, in order to obtain a method of analysis that can be conducted using living cells.

Method of analysis can be used to track the subcellular location and translocation of the complex protein-protein interaction through the use of permuteran luciferase or a fluorescent protein. In Fig. 4 is a diagrammatic representation of this example.

The present invention relates to methods, allowing us to set modulates if the connection interaction (i) a first protein, such as integrated in the membrane protein, for example, receptor, such as a transmembrane receptor, ii) a second protein, for example, intracellular molecule, another transmembrane protein, etc., for example, belonging to the family of krestinov. One of the methodologies includes joint transformation or transfection of cells that can be prokaryotes or eukaryotes, using the two constructions. The first structure includes a first nucleic acid encoding (a) a first protein, for example, a transmembrane receptor, and (b) a cleavage site for a protease, and (c) a second nucleic acid encoding a protein which activates a reporter. The second design includes (a) a nucleic acid encoding a second protein interaction is a journey of which the first protein is measured and (or) is determined, and (b) nucleic acid encoding a protease, a portion of the protease or a polypeptide with by activity, acting on the cleavage site of the first construction. In some embodiments, one or more of these structures may stably integrate into the cell.

Features of the example embodiment of the invention is graphically displayed in Fig. 1. In short, get a cell that expresses the first of the considered protein. Consider a protein may contain proteolytic part, or proteolytic part may be associated with complex upon binding or release of bound ligand. Inactivated enzyme binds to the peptide part, which is associated with the first of the considered protein in response to changes in the binding of the ligand. The proximity of the protease to inaktivirovannaja the enzyme in this example implementation can restore the activity of an enzyme, such as luciferase. The recovered activity affects the signal transmission of such protein-protein interactions.

In the example shown in Fig. 1, describes a transmembrane protein, an enzyme cleavage TEV, permuteran luciferase and luciferase substrate such as luciferin. In this example, as a protein "A" may be arrested. First consider the protein may be a GPCR. N - the C-terminal end of the luciferase you can reorganize and to associate with the site of cleavage of the TEV protease, to get inactivated, permuteran the luciferase. As shown in the figure, permuteran luciferase fused with β-arresting-2.

Protein And can merge with the protease. Protein can merge with inaktivirovannye permuteran protein which activates a reporter. Site recognition and cleavage of the protease (which is recognized by the protease, attached to the protein And injected into permuteran protein which activates a reporter. Protein a and protein To bring together, for example, the third molecule that modulates the interaction between protein a and protein C. Proteolysis permuteran inactivated protein which activates a reporter, under the action of nearby merged protease leads to the fusion of the two fragments permuteran protein which activates a reporter, in order to restore the activity of the active protein which activates a reporter. The activity of a protein which activates a reporter, can be evaluated using the appropriate reagents and instruments in the presence of a suitable reporter, for example, luciferin, using commercially available reagents and kits.

GPCR as protein A can merge with TEV protease. Alternatively, the GPCR may merge with permuteran protein which activates a reporter.

In Fig. 1 molecule that binds to GPCR causes interaction of β-arrestin with GPCR. Proteolysis of sitaramayya in the structure permuteran luciferase by TEV protease, which is associated with protein a or is close to it, enables the generation of fragments of the protein luciferase. Fragments restore active luciferase, which detects or presence of which make the conclusion on the basis of a suitable reporter luciferase activity, such as luciferin, in the cell or lysate.

This method can provide specific signals to receptor proteins, for example, a GPCR that may interact with G-protein or β-arresting.

The General method shown in Fig. 1, is applicable, generally, to a GPCR, as part of the beta arrestin is common. However, in practical applications of the present invention can use any pair of molecules that interact, communicate, associate among themselves, etc. or are assumed to interact, communicate, associate between themselves and other

In this example the method is used, the transmission signal of β-arrestin, which does not require any preliminary information on the specific binding of G-protein, as used analysis method is non-specific in relation to the GPCR or involved in G-protein. Therefore, the application of this analysis method, it is desirable for the orphan GPCR for which the unknown path of binding to G-protein. This method provides excellent the provides operational and physiologically meaningful results without transcription-mediated amplification, as suggested in the analysis, described in U.S. patent No. 7049076 (Lee et al.).

Materials and methods also allow monitoring of phenomena, independent of G-protein. In this case, β-arrestin you can mark permuteran protein which activates a reporter. The molecule, which is intended or likely interacts with β-arresting may be tagged with an appropriate protease, for example, a GPCR receptor, which shows a tendency to bind β-arrestin.

The present invention has advantages in comparison with the methods of analysis complementaly fragments of the enzyme (such as analysis of β-arrestin DiscoveRX PathHunter^TM), in which the interaction partners must remain connected or be around, in order to ensure the complementation of fragments of the enzyme. On the other hand, in this method of analysis after proteolysis due to proximity of reagents into active activator reporter, and for good informative samples does not require protein partners for cooperation remained bound.

In cell host may be injected nucleic acid encoding a first protein and other peptide components. Such engineering of cells are well known to experts in the field. Nucleic acid for different peptides can con trueroots in the form of single molecules or may be injected sequentially or in parallel. Some designs can be integrated into the chromosome of the host, for example, to ensure stable transfection, using materials and methods known to experts in the field.

In an alternative two of the studied protein can interact in the absence of ligand or test compound. The ligand or test compound can cause the dissociation of the two proteins change their conformation or any other way to weaken or inhibit their interaction. In this case, in the presence of a positive current test connection free, functionally active proteolytic enzyme in the cell decreases, which reduces proteolysis and registered drop in activity of a protein which activates a reporter.

In one example implementation arrestin acts as a second protein which binds to a transmembrane receptor in the presence of agonist; however, it should be understood that because the receptors are only one type of protein, the method of analysis does not depend on the use of molecules of the receptor, and the binding of the agonist is not the only interaction that may be involved here. Any protein that interacts with a second protein, meets, but of special interest Tran the membrane proteins due to their ability to cause a reaction cell, organ and tissue when exposed to a modulator, which provides integrated cell receptor in the active state of the receptor. In addition, the binding of agonist to the receptor is not the only link that can be analyzed. In this method of analysis can also be tested inverse agonists. You can install the antagonists themselves, but also to determine the relative effectiveness of different antagonists and (or) agonist in accordance with the present invention.

Below are other detailed information about the invention, including specific methods and techniques of preparation and use of the object.

As in the case of the method described herein, the products that are the distinguishing characteristics of the present invention are amenable to simple description. For example, in "design of three parts, i.e. constructs containing sequences that encode (i) a protein, (ii) a cleavage site; and (iii) a protein that activates the reporter protein can be, for example, intracellular protein or integrated into the membrane protein, such as a transmembrane receptor, for example, a member of the GPCR family. The cleavage site can be a hydrolyzable site, the hydrolysis can be performed when exposed to protease protein partner white is-protein interaction. Splitting can directly lead to the formation of the signal, or splitting can provide the reorganization of the protein which activates a reporter, in order to boost the signal from the other molecules. The third component may also be a protease or a polypeptide with by activity.

These sequences can be modified so that the C-terminal end of the proteins that they encode, entered into a more efficient and effective interaction with a second protein. Such modifications can include, for example, replacing the sequence encoding the C-terminal end of the protein, for example, GPCR, a region that encodes a C-terminal end of AVPR2, AGTRLI, F2PLI, CCR4, CXCR2/IL-8 and other Sequences of genes can be re-coded so as to optimize the transmission of the considered proteins in the cell host.

As a protein which activates a reporter, can be a protein that acts within the cytoplasm or organelles, such as nuclei, or there may be a molecule that activates a cascade of reactions leading to the reactions of the other protein. A qualified specialist in the field are well aware of these stages, as they are studied in detail cellular processes. For example, in the reporter enzyme can be activated translocation signals, such as serial is inost nuclear translocation. Specialists in the area of known sequence localization.

The second design, as described above, includes a region which encodes a protein that interacts with the first protein, resulting in a recorded phenomenon. Protein can be an activator, a competitor inhibitor, may provide a synergistic response, etc. or, in more General terms, to be a "modulator" of the first protein. As examples are the family members of arresting, especially if the first protein is a GPCR, but there may be other sequences encoding proteins, in particular, if the first protein is not a GPCR. The second of these two structures encodes a protease, a portion of the protease or a polypeptide with by activity, which are designed to break down activates the reporter protein encoded by the first design, in order to obtain activating a reporter protein that can directly or indirectly induce the detected signal.

While the above embodiments do not limit the scope of the invention as discussed in the following additional examples of implementation, for example, depending on the objectives protease may combine with protein a or protein Century

A host cell

Used herein, the terms "cell", "whether the Oia cells" and "cell culture" can be used interchangeably. The term "a host cell" may also refer to the original cell from which you can get the lysate. All these terms also apply to their offspring, which includes without exception, all subsequent generations. It should be understood that all progeny may be non-identical due to intentional or random mutation, selection or differentiation. A host cell may be constructed in such a way as to ensure the expression amenable to screening or selection marker or a reporter, providing a generated signal when exposed to activating the reporter protein first design, which is cleaved by the protease, forming part of a fused protein of the second design. For the introduction amenable to screening marker or reporter in the cell-master or system to be analyzed can be any of the methods.

For use as a host cell, there are numerous cell lines and cultures. For example, many of them can be obtained through the American type culture collection (ATCC) - the entity acting as an archive for living cultures and genetic materials. Professionals can determine the appropriate host based on the structure of the vector and the desired result. For example, to replicate many of vectors in a prokaryotic cell, the host can be entered n asmida or cosmid. The types of cells that are suitable for replication and / or expression vector, among others, include bacteria such as E. coli (such as E. coli strain RR1, E. coil LE392, E. coil B, E. coli X 1776 (ATCC No. 31537), E. coli W3110 (F^-lambda^-, prototroph, ATCC No. 273325), DH5α, JM109, and KC8), bacilli, for example Bacillus subtilis; and other enterobacteria, e.g., Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species, as well as a number of commercially available bacterial hosts, e.g., competent cells SURE^®and cells SOLOPACK^TMGold (STRATAGENE^®, La Jolla). In some embodiments as host cells for phage can be used in bacterial cells such as E. coli LE392.

Examples of eukaryotic host cells for replication and / or expression vector, among others, are HeLa, NIH3T3, Jurkat, 293 (HEK), COS, CHO, Saos, and PC12. Also suitable and other cells, such as yeast cells or insect cells, such as Sf9 cells. Qualified specialists in the field shall have the discretion to choose the cell host, which they intend to use for the intended purpose. Qualified professionals are available and there are many host cells among different cell types and species of organisms.

Similarly, in combination with a eukaryotic or prokaryotic cell host can be used as a viral vector (that is the number of phage), especially with such a cell, which allows the replication or expression vector. A host cell does not necessarily apply to immortalizing cell line. A host cell may be derived from stem cell cultures or cultures of primary cells, e.g. hematopoietic stem cells, vascular, epithelial cells, smooth muscle, spleen, T cells, b cells, monocytes, etc. a host Cell may be transgenic, for example, containing genetic material from another organism. Cells that cannot be used in the method of Lee et al., suitable for analysis in the framework of the present invention, since in this case there is no need for active transcription. For example, in the present invention can be used in non-nuclear cells, such as erythrocytes or platelets.

In the context of the method it is assumed that the term "a host cell" includes an artificial environment and particles, such as liposomes and virus-like particles. Such structures are often mimic or simulate the cell or its part by creating a shell surrounding an empty space, which is fully or partially separately from the external environment by a film, membrane or other structure. As already noted, such an artificial environment, and the particles are liposomes, spiralia the data units, virus-like particles, viruses and other

Proteins

In the present invention it is proposed to use any two of the protein for which it is known or assumed physical interaction. In some embodiments, the proteins will exist or will be constructed in the form of a fused protein, the first protein fused with latent or inaktivirovannye a polypeptide that activates the reporter, and the second protein is fused with a protease that recognizes the cleavage site in the first fused protein, the cleavage which releases the activating reporter polypeptide or provides its activity.

With regard to the first of the studied protein, the first protein may be, for example, found in nature is integrated into the membrane protein or protein, designed to ensure its integration into the membrane. For example, the first protein may be a transmembrane receptor, such as a GPCR, or any other question transmembrane receptor, including, among others, the receptor tyrosinekinase, receptor serine/treoninove kinases, receptors, cytokines, etc. in Addition, since it is well known that part of the protein will function in exactly the same way as the first full length protein, such an active part of the first protein, for example, the extracellular domain and the transmembrane domain, include the camping in the coverage of protein determination in this document.

As will be obvious to a qualified specialist in the field, the present invention can be used to analyze interactions with any protein, and its coverage is not limited to the analysis of integrated membrane receptors, such as GPCR. For example, the activity of other classes of transmembrane receptors, including, among others, the receptor tyrosinekinase (RTK), for example, IGF1R, for example, receptor, epidermal growth factor (EGFR), ErbB2/HER2/Neu or related RTK, receptor serine/treoninove kinases, for example, transforming growth factor-β (TGFβ), activin or receptor bone morphogenetic protein (BMP); cytokine receptors, such as receptor family of interferons for interleukin, erythropoietin, G-CSF, GM-CSF or tumor necrosis factor (TNF)receptors leptin; and other receptors that are not necessarily integrated into the membrane in the normal state, for example, estrogen receptor-1 (ESR1) and estrogen receptor-2 (ESR2). In each case, this method may provide for the transfection of cells modified polynucleotides receptor, which determines the expression of a chimeric or fused protein, including the considered receptor cleavage site of the protease and of the polypeptide that activates the reporter. The cell may jointly transactivates second polynucleotide, for example, a vector that is what determines the expression of a chimeric or fused protein, including interacting protein, merged with a protease that recognizes and cleaves the cleavage site of the first protein. The first and second polynucleotide can be combined in a single molecule, thereby it is possible to avoid co-transfection. In the case of RTK, e.g., EGFR, such interacting protein may consist of SH2 (Src domain homolog-2)containing the polypeptide, such as phospholipase C (PLC), or domain Src homolog-2 containing transforming protein 1 (SHC1). In the case of receptor serine/treoninove kinases, such as receptor TGFβ, activin and BMP, so interacting polypeptide may be a Smad protein or part thereof. In the case of receptors of cytokines, for example, receptors, interferon α, interferon β or interferon γ, such interacting protein may be a signal transmitter and activator of transcription (STAT), for example, among others, Stat1 or Stat2; or proteins Janus kinase (JAK), Jak1, Jak2 or Tyk2; or their parts and other Transfectional cell can contain a reporter, which operates activating the reporter protein. Then conduct the analysis, which transfection cells treated with test compound for a certain period of time, and upon completion of the testing period measure the activity of the reporter. If the test compound activates the considered receptor occurs trims the regulation of interactions between the observed and receptor interacting protein which leads to the cleavage site of the protease and activation of protein which activates a reporter that, in turn, leads to a detectable change or increase the activity of the reporter.

The other possible pairs of proteins are antibody-antigen, enzyme-substrate, dimerizes proteins, components of the cascade of the transmit signal component(s) of complex structures, such as ribosomes or virus, interacting at the intercellular level of molecules in different cells, for example, the antigen-containing cell and immune cell response, such as T-cell, b-cell, NK cell, dendritic cell, monocyte, macrophage, etc. as well as other pairs of proteins known to experts in the field. Determination of protease and protein to the site of the protease recognition are used interchangeably with respect to each of the proteins, for example, a or b, with which each of them is linked or associated.

Reporters

As a reporter can be any molecule that alters the structure or function in response to the impact of active molecules that activate the reporter, and allows to obtain the detected signal, or can easily be monitored to track such changes. It is expected that these terms will be used freely. Protein which activates a reporter, after its activation (or inactivation of some possible p is imarah implementation) causes detective change in the reporter molecule. Registration of this change is used to determine, for example, modulated test whether the connection of protein-protein interactions. In the event of a detected signal can be used by other proteins that activate the reporter, which are not enzymes. Therefore can be used a known protein which activates a reporter, for example, galactosidase, peroxidase, luciferase, etc. Can be used known reporters, for example, substrates galactosidase, peroxidase substrates, the substrate of luciferase, GFP, etc.

Protease and cleavage sites

Proteases belong to the well-characterized enzymes that break down other proteins in a particular site. One of the families, a family of serine/treoninove proteases, provides splitting at the location of the residues serine and / or threonine. Other proteases include cysteine or thiol-proteases, aspartate proteases, metalloproteases, amino peptidases, di - and tripeptides, carboxypeptidase and paticipated. The choice of protease remains a qualified expert in the field, and this choice should not be limited to the molecules described herein. It is well known that enzymes contain a catalytic domains, and these domains can be used instead of proteases full length. They also fall within the scope of coverage of the present invention. One specific implementation is a protease nuclear inclusion virus engraving tobacco And (TEV) or its active part. Can also be used in other specific cleavage sites for proteases that it will be obvious for a skilled specialist in the field.

Modification of proteins

In some embodiments of the present method of analysis the first protein may be modified to enhance its binding to interacting proteins. For example, it is known that some GPCR associated arrestin more stable, or a higher affinity with the stimulating ligand, and is more effective interaction mediated by separate domains, for example, clusters of residues serine and threonine in the C-terminal end (Oakley et al,J. Biol. Chem., 274:32248-32257, 1999 and Oakley et al.,J. Biol. Chem., 276:19452-19460, 2001). With this example it becomes obvious that the sequence encoding the receptor can be modified in order to increase the affinity of integrated membrane protein such as a receptor for the protein to which it binds. Examples of such changes include modification of the C-terminal region is integrated into the membrane protein, for example, 7TMR, which may include the replacement part on the corresponding area of another receptor, which has more than the high binding affinity to the protein, but does not affect the function of the receptor binding.

In addition or alternatively, the second protein may be modified in order to increase the interaction with the first protein. For example, the method of analysis may include point mutations, the results of the processing or other variants of the second protein, for example, arrestin, which are known to be more stable or independent of phosphorylation way bind occupied by the agonist of the GPCR receptors (Kovoor et al.,J. Biol. Chem.,274:6831-6834, 1999). Such changes can be made using methods known to experts in the field.

Format analysis

In several embodiments of the present invention offers a simple way to assess the interaction of two proteins in their expression in the same cell, particle, or the reaction mixture. The first structure may include a sequence encoding a first polypeptide linked with polynucleotides coding for the cleavage site of the protease, part of the protease or a polypeptide with the activity of proteases, which, in turn, is in concatenation with polynucleotides, encoding the reporter enzyme. The term "concatenation" describes a situation in which these sequences are merged so that a single, intact open reading frame, which the traveler can be translated into a single polypeptide, containing all of the elements. They can, but not necessary, to divide the extension nucleotides, which can encode or not to encode other proteins or peptides. The second design, introduced in the recombinant cell may contain polynucleotide that encodes the second protein, and protease, part of the protease or a polypeptide encoding the protease activity. Taken together, these elements form the basic format analysis method in combination with a potential agent, whose influence on the interaction of the target protein investigated in this case.

This invention can also be used to analyze multiple integrated into the membrane proteins simultaneously, for example, receptors, using a variety of reporters, each of which is stimulated by activation of protein, for example, proteins of the classes described in this document. For example, this can be achieved by mixing cells, transfection different structures of receptors and various proteins that activate the reporter, or by merging different enzymes for each test receptor, and measuring the activity of each reporter gene in the processing of the test compound (compounds). For example, it may be desirable to determine activates if the first molecule receptor, as well as to install, ledue what to expect side effects as a result of interaction with a second receptor. In this case, for example, to use the first line of cells encoding a first receptor and a first protein which activates a reporter, for example, lacZ, and a second line of cells encoding a second protein that activates the receptor, and the second reporter, for example, GFP. In these circumstances, GFP can be permuteran in accordance with the practice of the present invention. You can mix the two cell lines, add the considered connection and monitor the positive effect of one of them with no effect on another.

The invention in alternative formats refers to methods of analysis, where we study the one pair of interacting proteins, as well as to cases which are herein referred to as "multiplex" methods of analysis. Such analysis techniques can be used in various ways, but in all cases tested simultaneously more than one pair of proteins. This can be achieved, for example, through the use of multiple samples of cells, each of which has been transformed or transfection for testing each interacting pair of proteins. Various transformed cells can be combined and run simultaneously in a single receiver, or each of the different types of transformant can be placed in different wells, and then test. Alternatively, the cell can be manipulated in order to work with many labeled the first protein, for example, transmembrane proteins, to determine whether the ligand or potential molecule to activate more than one receptor.

Cells used in multiplex assays described herein can be the same, but not necessarily. Similarly, the reporter system used in each sample, may be the same, but not necessarily. After the sample or the sample is placed in the receivers, for example, in the wells of the microplate can be screening one or more compounds relative (if possible) to the set of interacting pairs of proteins placed in the receivers.

In Fig. 10 provides an example of typical results obtained during this analysis. At low or high concentrations (in the matter is whether the modulation of inhibitory or activating) the test compound may not have any effect. Decreasing or increasing the concentration of the modulating effect may vary. To assess modulation can be used curve of the response to the dose shown in Fig. 10. You can also calculate the values of a point. For example, a single point may be a predetermined value that is different from the reference or background, which is often determined on a statistical basis through the m data accumulation or analysis of several samples from normal subjects, in order to obtain a mean value for samples with standard errors and deviations. In quality pre-determined value difference can be a constant. Typically, the ratio is used, for example, at least 10% of the control values, but frequently used values that are multiples of the control value, for example, about 1,5, 2, 2,5, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more (or return them to the value), multiplied by the reference value, which can be pre-installed in a different cycle analysis. Predetermined limit value, indicating that the modulation is calculated by a qualified technician according to standard methods taking into account the compensation of errors type 1 and 2 depending on the capabilities or needs of the situation.

Test kits

Any of the compositions described herein, and their combinations can be supplied in the form of a test kit. Such kits will include suitable capacity (capacity), one or more components, for example, vectors or cells of the present invention, and any additional agents that can be used in accordance with the present invention.

The kits can include one or more appropriately selected aliquot of the compositions of the present invention. Kit components can lithophane in the form of aqueous solutions or in liofilizovannyh form or in the form of a concentrate in a suitable for the solute to the solvent. Capacity (capacity) of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container, into which you can place the component or in which he is placed, preferably, in properly selected aliquot. If the dataset for analysis has several components, then this set typically will also include second, third, etc. capacities, which can be separately placed additional components. While various combinations of components can be placed in one container, such as a bottle. In addition, there may be attached a suitable solvent. The kits of the present invention also will typically include a means to ensure the immobility of the containers with reagents in the packaging for commercial distribution. Such means may be made of blow forming containers made of plastic or Styrofoam, which stores the bottles along with printed instructions.

If the components of the kit are supplied in the form of a single (or multiple liquid solutions, this solution can be an aqueous solution, for example, is most often used sterile aqueous solution. However, the components of the test kit can be supplied in the form of ugogo powder (powder) or deposited on a solid substrate. If the reagents and / or components are supplied in the form of a dry powder, the powder can be dissolved by adding a suitable solvent. It is assumed that solvents such as sterile water or suitable saline solution or a buffer can also be supplied in other capacity.

EXAMPLES

Specific examples of the implementation of describing the present invention, are described below in the examples, but they should not be construed as limiting the scope of the invention.

Example 1

In Fig. 1 is an example implementation that includes permuteran inactivated luciferase activity, which is restored when the effects of TEV protease at contained in the molecule site recognition TEV protease. As shown, the first protein fused with a protease. Example 1 involves the ability to apply the activity of TEV protease for recovery permuteran luciferase serving as the second protein. The second protein, as shown, merge with inaktivirovannye permuteran protein which activates a reporter luciferase. Site recognition and cleavage of the protease (which is recognized by the protease fused to the protein) is introduced into permuteran protein which activates a reporter. The first and second proteins are brought together by a third molecule is, which modulates the interaction between the first and second proteins. Proteolysis permuteran inactivated protein which activates a reporter, under the action of nearby merged protease leads to cleavage with the formation of two fragments permuteran protein which activates a reporter, in order to regenerate the active protein which activates a reporter. The activity of a protein which activates a reporter, can be evaluated using the appropriate reagents and instruments.

Permuteran the luciferase was constructed by reorganizing the N-terminal amino acids 2 through 233 of the Firefly luciferase and the C-terminal amino acids 234 to 550 in reverse order, interrupted by the website recognition TEV protease, ENLYFQX (SEQ ID NO:3). Cleavage at this site leads to the restoration activity permuteran luciferase. At position X can be any amino acid that determines the affinity recognition TEV protease and cleavage efficiency. It was shown that the change in X allows you to modulate the enzymatic kinetics TEV. A similar substitution of amino acids at other sites in the sequence recognition can also change the kinetics. Modulation of the kinetics is useful for optimization, for example, the incubation time in the screening process and background activity, which affects attributed the s/n ratio. Permuteran the luciferase (luc234X233, where X is a specific amino acid in the N-terminal end of the heptapeptide cleavage site TEV, SEQ ID NO:3) was then merged with the C-terminal end of the GPCR, ADR 2, in order to obtain GPCR-permuteran the luciferase, ADR 2-luc234X233, plasmid expression.

Example 2

Human fused plasmid β-arrestin-2-TEV constructed by merging protease And virus engraving tobacco with the C-terminal end of β-arrestin-2. All DNA fragments were obtained by PCR using appropriate matrices. Fused genes GPCR-luc234X233 was subcloned into the pcDNA3.1(+) in the presence of the marker selection neomycin (Invitrogen), and fused genes Arr-TEV was subcloned into the pcDNA3.1(+) marker selection zeocin (Invitrogen No. cat. 43-0018).

Example 3

Cells CHO-K1 was transfectional together with ADR 2-luc234R233 (example 1) and the plasmid Arr-TEV (example 2) using the appropriate kits for standard transfection. Forty-eight hours after transfection cells within 2 hours were treated in the presence or absence of 10 μm ADR 2 agonist, isoproterenol to the cells was added Bright-GLO^TMor Steady-GLO^TM(Promega) and using the appropriate tablet reader luminescence was detected relative luminescence lysates. In the presence of isoproterenol was observed more than three-fold increase online is newnote luminescence.

In Fig. 5 presents data for the expression of GPCR/permuteran luciferase in the presence and in the absence of Arr2-TEVp.When registering data shown in the graph, constructs were introduced into cells, but transfection cells were not subjected to any modulator. Therefore, evidence suggests that if the cleavage site contains serine, recorded some spontaneous activity, but, essentially, the background noise does not occur if X is R or V. As noted in Fig. 6, when the cells expressing the cleavage site with R or V were subjected to exposure to agonist, was observed response.In Fig. 7 shows dose-dependent effect of luciferase activity in cells that are intermediate or stably Express a GPCR-luc234V233 and (or) Arr-TEV.

Example 4

In Fig. 8 provides evidence that 5-hour or 1-hour incubation period is sufficient for the analysis of protein-protein interactions. Dose-dependent effect clearly presented.

Plasmid expression fused gene ADR 2-TEV constructed by merging protease And virus engraving tobacco with the C-terminal end ADR 2 and the introduction of the fused gene into pcDNA3.1(+) marker selection zeocin (Invitrogen No. cat. 43-0018). All DNA fragments were obtained by PCR using appropriate matrices, known to specialists in the sphere of the.

Plasmid expression fused gene β-arrestin-2-permuteran luciferase (Arr-luc234X233) constructed by merging permuteran luciferase luc234X233 with the C-terminal end of β-arrestin-2. The cleavage site of the protease TEV is ENLYFQ/X (Rachel B. Kapust, et al.Biochemical &Biophysical Research Communications, 294 (2002) 949-955), where E and Q are usually invariant and where X can be any amino acid, though most of this site contains amino acids G and S. the Cleavage occurs between residues Q and X. X can determine the efficiency of cleavage. In some embodiments, the cleavage site of the protease TEV included in the structure permuteran luciferase. The background and signal-to-noise ratio can be improved by using simple standard experiments. For example, it was found that the use of valine instead of the glycine site of hydrolysis of X for TEV in some applications reduces the background. Composed of fused gene cloned in pcDNA3.1(+) marker selection neomycin (Invitrogen).

The HEK293 cells were transfectional together with plasmids ADR 2-TEV and Arr-luc234V233, where the sequence recognition TEV is ENLYFQV (SEQ ID NO:12), using the appropriate standard kits for transfection. Forty-eight hours after transfection the cells for 1 and 5 hours were treated with various concentrations of ADR 2 agonist, isoproterenol, the cell is m added Bright-GLO ^TM(Promega) and using EnVison II^TMregistered relative luminescence particles. After 1 and 5 hours after incubation with isoproterenol observed a dose-dependent intensity of luminescence.

Example 5

In Fig. 9 in the left panel presents induced ligand luciferase activity in HEK293 cells, stably expressing the fused protein Arr-luc234V233 and intermediate expressing the fused protein ADR 2-TEV. In the right pane displayed stable expression design arrestin - protein that activates a reporter, and intermediate expression merged 7TMR-protease in CHO cells.

A stable cell line expressing GPCR-luc234R233 or Arr-TEV, obtained for cells HEK293 or CHO. For transfection used twenty ng/well of each DNA in 12-hole tablet with lipofectamine (Invitrogen) for HEK293 cells and TranIT-CHO.

In the analysis per-luc standard used 384-well format plates. Other formats tablets was considered acceptable formats. The CHO cells, stably expressing GPCR-luc234R233 or Arr-TEV, inflicted on 10,000 cells per well on the surface of the treated tissue cultures in 384-well white analytical tablet (Becton Dickinson). The next day cells were treated with agonist concentrations from 10 μm to 0.7 PM (in serial dilutions of 3:1, conducted in free serum cellular environment). For the serenia activity of luciferase used the system for analysis of luciferase Steady-Glo (Promega). After 2 hours of treatment with agonist medium was aspirated and each well was added 25 μl of analytical reagent luciferase. The value of relative luminescence units (RLU) were determined using EnVision, universal reader produced by Perkin Elmer. The data displayed in a graph and analyzed using the software PRISM.

The HEK293 cells, stably expressing Arr-luc234V233, made by selection for resistance to neomycin. Resistant to neomycin gene is represented in the vector pcDNA3.1 plasmid expression Arr-luc234V233.

Example 6

On the right panel of Fig. 9 provides data for dose-dependent effect of isoproterenol in line SNO.

A stable cell line expressing GPCR-luc234R233 or Arr-TEV, obtained in CHO cells. One μg of each DNA was selected for transfection in each well of a 12-hole tablet using the kit for transfection TransfectIT-CHO (cool simple point pointers rubber Bio, Madison, Wisconsin). The transfectants were collected single colonies during breeding using neomycin or neomycin.

Stably expressing Arr-luc234V233 cells were transfectional the plasmid ADR 2-TEV using standard kits for transfection. Intermediate expressing ADR 2-TEV and stably expressing Arr-luc-234V233 cells were incubated with isoproterenol for two hours and then the cells were added to the reagent BrightGLO luciferase ^TM. Dose-dependent effect of luciferase was detected by EnVison II.

Example 7

In Fig. 10 shows the estimation results of the analysis of GPCR Per-Luc with an agonist, partial agonist, antagonist, and nonspecific responses of the endogenous receptor.

Stably expressing Arr-luc234V233 HEK293 cells were transfectional the plasmid ADR 2-TEV using the transfection reagent of lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection cells were incubated with different concentrations of a known agonist, isoproterenol; partial agonist BRL37344 (Sigma-Aldrich); antagonist ICI118551 (ICI); antagonist ICI118551 with 200 nm isoproterenol; and agonist S1P (sphingosine-1-phosphate) endogenous EDG receptors for HEK293 cells within two hours, and cells were added reagent luciferase Bright-GLO^TM. Dose-dependent luciferase activity was detected by EnVison II, as shown in Fig. 10.

The values of EC50 and IC50 obtained in the analysis were similar in magnitude to the values observed in the FLIPR assays and camp. Endogenous receptor EDG in HEK293 cells and its ligand S1P did not affect the luciferase activity, whereas in other methods of analysis, e.g., FLIPR and camp, was actually recorded positive signals. The agonist isoproterenol ensured the generation of a response. For partial agonist BRL37344 noted on tpny response. Antagonist ICI18551 inhibit isoproterenol, but by itself did not show activity. Therefore, the proposed analysis method is specific and, as shown in Fig. 10 (along with other data for comparison), is characterized by less frequent and less intense false signals.

Example 8

In Fig. 14 shows an example in which permuteran the luciferase was constructed by cloning the N-terminal amino acids 2 through 456 luciferase Firefly for the C-terminal amino acids 234 to 550, with site recognition TEV protease, ENLYFQX, where instead of X used V. Permuteran the luciferase (luc234V456) was merged with the C-terminal end of the GPCR, ADR 2, in order to get the design of GPCR-permuteran luciferase, plasmid expression ADR 2-luc234V456.

All DNA fragments were obtained by PCR using appropriate matrices. Fused genes ADR 2-luc234V456 cloned in pcDNA3.1(+) marker selection neomycin (Invitrogen).

Cells CHO-K1 was transfectional together with ADR 2-luc234V456 and plasmids Arr-TEV using the appropriate kits for standard transfection. Forty-eight hours after transfection cells within 2 hours were treated in the presence or absence of 10 μm ADR 2 agonist, isoproterenol to the cells was added Bright-GLO^TMor Steady-GLO^TM(Promega) and using the appropriate Board is and reader luminescence was detected relative luminescence. Registered restored luciferase activity in response to different doses of isoproterenol.

The HEK293 cells, stably expressing Arr-luc234V233, made by selection for resistance to neomycin. Resistant to neomycin gene is represented in the vector pcDNA3 plasmid expression Arr-luc234V233. Registered luciferase activity in response to exposure to agonist.

Example 9

In Fig. 13 shows the dependence of the dose for inverse agonist V2.

For this example, HEK293 cells, stably expressing Arr-luc234V233, transfectional the plasmid V2-TEV using the transfection reagent of lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection cells within two hours were incubated with various concentrations of compounds SR121463 (Sanofi Recherché, Toulouse, France), which is considered an antagonist, using standard methods of analysis. To the cells was added to the reagent luciferase Bright-GLO^TM. Dose-dependent luciferase activity was detected by EnVison II. That is, increasing levels of luminescence intensity was observed with increasing amounts of SR121463, which can more accurately be attributed to inverse agonists.

In this analysis, inverse agonist behaved as an agonist, like other inverse agonists. It is known that the inverse agonist able to block transmission of the signal G-b is the left main coronary artery for V2, at the same time stimulating β-arrestin-mediated activation of MAPK (Azzi et al., PNAS, 2003,100:11406-11411). Therefore, the analysis method of the present invention can indicate distinctive active conformation for receptor associated G-proteins.

On the contrary, in classical systems analysis reverse GPCR agonists behave as antagonists. This is due to the fact that inverse agonists, apparently, are associated with inactive GPCR conformations for signal transmission G-protein and stabilize them. However, some inverse agonists stabilize the inactive form of the GPCR signal G-protein, as well as contribute to the capture of β-arrestin in GPCR to activate the transmission path of the signal through β-arrestin.

Example 10

In Fig. 6 shows the luciferase activity induced by the agonist.

In this example, permuteran the luciferase was constructed by reorganizing the N-terminal amino acids 2 through 233 of the Firefly luciferase and the C-terminal amino acids 234 to 550 in reverse order, interrupted by the website recognition TEV protease, ENLYFQX. At position X can be any amino acid. It is known that amino acids in this position determine the affinity recognition of TEV protease and cleavage efficiency. Permuteran luciferase (luc234X233) was then fused with the C-terminal end of the GPCR, that is, DRB2, to get GPCR-permuteran the luciferase, i.e. a plasmid expression ADR 2-luc234X233.

Human fused plasmid β-arrestin-2-TEV designed by merging protease And virus engraving tobacco with the C-terminal end of β-arrestin-2. The DNA fragments were obtained by PCR using appropriate matrices. Fused genes GPCR-luc234X233 was subcloned into the pcDNA3.1(+) in the presence of the marker selection neomycin (Invitrogen), and fused gene Arr-TEV was subcloned into the pcDNA3.1(+) marker selection zeocin (Invitrogen No. cat. 43-0018).

Cells CHO-K1 was transfectional together with ADR 2-luc234R233 and plasmids Arr-TEV using sets, suitable for standard transfection. Forty-eight hours after transfection cells within 2 hours were treated in the presence or absence of 10 μm ADR 2 agonist, isoproterenol to the cells was added Bright-GLO^TMor Steady-GLO^TM(Promega) and using the appropriate tablet reader luminescence was detected relative luminescence. In the presence of isoproterenol was observed by more than three-fold increase in luminescence intensity.

Example 11

In Fig. 12 shows the results of the analysis for agonists, antagonists and inverse agonists using the analysis methods of the present invention (left panel) and other methods of analysis (right panel). In two of the graphs displays the Agen differentiation and the effects of counter-agonist, antagonist and inverse agonist. The proposed analysis method provides a high specific activity.

Example 12

In Fig. 7 shows CHO cells with ADR 2-permuteran luciferase and Arr-TEV.

For the data in the left panel of ADR 2-luc234V233 designed in such a way as to contain the site of TEV recognition, ENLYFQV. Cells CHO-K1 was transfectional together with ADR 2-luc234V233 and plasmids Arr-TEV using the appropriate kits for standard transfection. Forty-eight hours after transfection cells within 2 hours was treated with various concentrations of ADR 2 agonist, isoproterenol to the cells was added Bright-GLO^TMor Steady-GLO^TM(Promega) and using the appropriate tablet reader luminescence was detected relative luminescence.

Example 13

In the right panel of Fig. 7 summarizes data using stable transfection cells with different cleavage sites. These results are similar to the one shown in the left panel the results. Thus, two designs of GPCR-luciferase with different cleavage sites responded to agonist.

Example 14

In Fig. 6 shows data on agonist-induced activity signal when the mapping X = R and X = V. the Results are similar, which indicates the possibility of free variation X.

This item is the iMER permuteran the luciferase designed by reorganizing the N-terminal amino acids 2 through 233 of the Firefly luciferase and the C-terminal amino acids 233 to 550 in reverse order interrupted by the website recognition protease TEV, ENLYFQ/x position X can be any amino acid that determines the affinity recognition TEV protease and cleavage efficiency. Shown V and R. Permuteran the luciferase (luc234X233) then merged with the C-terminal end of the GPCR, i.e. ADR 2 to get GPCR-permuteran the luciferase, i.e. a plasmid expression ADR 2-luc234X233.

In this example, human fused plasmid β-arrestin-2-TEV designed by merging protease And virus engraving tobacco with the C-terminal end of β-arrestin-2. All DNA fragments were obtained by PCR using appropriate matrices. Fused genes GPCR-luc234X233 was subcloned into the pcDNA3.1(+) in the presence of the marker selection neomycin (Invitrogen), and fused genes Arr-TEV was subcloned into the pcDNA3.1(+) marker selection zeocin (Invitrogen No. cat. 43-0018).

Cells CHO-K1 was transfectional together with ADR 2-luc234R233 and plasmids Arr-TEV using the appropriate kits for standard transfection. 48 hours after transfection cells within 2 hours were treated in the presence or absence of 10 μm agonist ADR 2, the cells were added Bright-GLO^TMor Steady-GLO^TM(Promega) and using the appropriate tablet reader luminescence was detected relative luminescence particles. In the presence of isoproterenol was observed by more than three-fold increase in levels online is newnote luminescence.

Example 15

In the left panel of Fig. 11 shows the results for agonist 8-AVP in cells that provide intermediate expression V2-TEV.

In this example, HEK293 cells, stably expressing Arr-luc234V233, transfectional the plasmid V2-TEV using the transfection reagent of lipofectamine 2000 (Invitrogen). After 48 hours cells were incubated for two hours with different concentrations of the agonist 8-AVP (Arg⁸vasopressin, a known agonist of the receptors of vasopressin V2), and the cells were added to the reagent luciferase Bright-GLO^TM. Dose-dependent luciferase activity was detected by EnVison II.

Example 16

The HEK293 cells, stably expressing Arr-luc234V233, transfectional the plasmid V2-TEV using the transfection reagent of lipofectamine 2000 (Invitrogen). After 48 hours cells were incubated for two hours with different concentrations inverse agonist, and the cells were added to the reagent luciferase Bright-GLO^TM. Dose-dependent luciferase activity was detected by EnVison II.

In this method of analysis inverse agonist behaved as an agonist. It is known that some inverse agonists can block transmission of the signal G protein for V2, but stimulate β-arrestin-mediated activation of MAPK (Azzi et al.,PNAS, 2003,100:11406-11411). Therefore, this analysis method is able to assess ex is considerably active conformation for receptor associated with G-proteins.

Example 17

On the right panel of Fig. 11 displays dose-dependent luciferase activity in the feedback inverse agonist V2 by facilitating the interaction of β-arrestin with the V2 receptor.

In this example, HEK293 cells, stably expressing Arr-luc234V233, transfectional the plasmid V2-TEV using the transfection reagent of lipofectamine 2000 (Invitrogen). After 48 hours cells were incubated for two hours with different concentrations inverse agonist, and the cells were added to the reagent luciferase Bright-GLO^TM. Dose-dependent luciferase activity was detected by EnVison II.

Other distinctive features of the present invention will be apparent to the skilled expert and need not be repeated herein. A qualified specialist in the field can make various modifications without affecting the main idea and scope of the present invention.

All references cited in this document by reference fully incorporated in this document.

1. The method of identifying compounds modulating protein-protein interaction between the first protein and the second protein, which includes:
i) providing a first protein that is attached to the split and reorganized protein that activates R the porter, where a protein which activates a reporter contains the cleavage site by the protease, which is located between the two parts of the protein which activates a reporter, where these two parts of the protein which activates a reporter, are reorganized;
ii) providing a second protein that is attached to the protease where the specified protease capable of cleavage at the cleavage site in the specified protein which activates a reporter that leads to the reorganization of these two parts of the protein which activates a reporter than this protein is activated, activating the reporter; and where the Association of the first protein with a second protein leads to protein which activates a reporter, protease;
iii) providing a reporter, a signal which changes the activity of the specified protein which activates a reporter;
iv) obespechenie test compounds;
v) providing the possibility of splitting the specified cleavage site indicated by the protease; and
vi) monitoring signal;
moreover, changes in said signal indicates the presence of the indicated protein-protein interactions.

2. The method according to claim 1, where at least one of the first protein and the second protein is a protein associated with the membrane.

3. The method according to claim 1, where at least one of the first protein and the second protein is a cytoplasmic protein.

4. The method according to claim 1, where at least one of the first protein and the second protein is modified, with the aim of increasing its affinity binding to another protein.

5. The method according to claim 1, where the first protein selected from the group consisting of: receptor associated G-protein, β-adrenergic receptor, receptor arginine-vasopressin-2 receptor serotonin-1a, muscarinic acetylcholine receptor m2, a receptor of the chemokine-5 receptor dopamine D2, Kappa-opioid receptor α1a-adrenergic receptor, receptor insulin-like growth factor-1, estrogen receptor-1, estrogen receptor-2, frizzled receptor, the receptor for epidermal growth factor receptor tyrosine kinase, receptor serine/trionychinae, receptor transforming factor growth-β, activin, receptor bone morphogenetic protein receptor of the cytokine, interferon receptor, receptor interleukin, erythropoietin receptor, the receptor of tumor necrosis factor, leptin receptor, receptor-stimulating factor granulocyte colony, and receptor-stimulating factor granulocyte-macrophage colony.

6. The method according to claim 1, where the second protein selected from the group consisting of arrestin and binding protein Dishevelled.

7. The method according to claim 1, where the protease is selected from the group consisting of: protease nuclear inclusion virus engraving tobacco A (TEV), ante is akinosi, protease factor XA, thrombin, a protease PureAct™, protease Clean Cut™, protease PreScission™, serine/ser / thr protease, thiol protease, aspartate proteases, metalloproteases, amino peptidases, dipeptidase, tripeptides, carboxypeptidase and paticipated.

8. The method according to claim 1, where the protein which activates a reporter, is self-activating.

9. The method according to claim 1, where the protein which activates a reporter is a reporter.

10. The method according to claim 1, where the protein that activates the reporter is an enzyme.

11. The method according to claim 1, where the protein which activates a reporter, is a protein that causes a change in fluorescence of the reporter.

12. The method according to claim 1, where the fluorescent protein is a reporter.

13. The method according to claim 1, where the protein which activates a reporter selected from the group consisting of luciferase, Gaussia luciferase, Renilla luciferase, a fluorescent protein, green fluorescent protein, protein DsRed, peroxidase, β-galactosidase, β-lactamase.

14. The method according to claim 1, where the first protein forms a fused protein with a protein which activates a reporter.

15. The method according to claim 1, where the reporter is selected from the group consisting of: substrate galactosidase, peroxidase substrate, the substrate of luciferase and luciferin.

16. The method according to claim 1, where the signal from the reporter selected from the group consisting of luminescence and color change.

17. The method according to claim 1, where the protein-protein interaction requires the translocation of the first protein or the second protein in the cellular compartment or organelle.

18. The method according to claim 1, where the method further includes providing molecules known that it modulates the specified protein-protein interactions, where the test compound modulates the interaction of the specified molecule with the specified protein-protein interaction.

19. Analysis system, including:
i) a first protein, attached to a split and reorganized protein which activates a reporter, where a protein which activates a reporter contains the cleavage site by the protease, which is located between the two parts of the protein which activates a reporter, where these two parts of the protein which activates a reporter, are reorganized;
ii) a second protein, attached to the protease where the specified protease capable of cleavage at the cleavage site in the specified protein which activates a reporter that leads to the reorganization of these two parts of the protein which activates a reporter than this protein is activated, activating the reporter; and
iii) the reporter, the signal of which changes due to the activity of the specified protein which activates a reporter;
where the Association of the specified first protein with a second specified protein leads to the splitting of the specified protein which activates a reporter, the protease.

20. The analysis system according to claim 19, where at least one of the first protein and the second protein is the Wallpaper protein, associated with the membrane.

21. The analysis system according to claim 19, where at least one of the first protein and the second protein is a cytoplasmic protein.

22. The analysis system according to claim 19, where at least one of the first protein and the second protein is modified, with the aim of increasing its affinity binding to another protein.

23. The analysis system according to claim 19, where the first protein selected from the group consisting of: receptor associated G-protein, β-adrenergic receptor, receptor arginine-vasopressin-2 receptor serotonin-1a, muscarinic acetylcholine receptor m2, a receptor of the chemokine-5 receptor dopamine D2, Kappa-opioid receptor, α1-adrenergic receptor, receptor insulin-like growth factor-1, estrogen receptor-1, estrogen receptor-2, frizzled receptor, the receptor for epidermal growth factor receptor tyrosine kinase, receptor serine/trionychinae, receptor transforming growth factor-β, activin, receptor bone morphogenetic protein receptor of the cytokine, interferon receptor, receptor interleukin, erythropoietin receptor, the receptor of tumor necrosis factor, leptin receptor, receptor-stimulating factor granulocyte colony, and receptor-stimulating factor granulocyte-macrophage colony.

24. The analysis system according to claim 19, where the second protein vibrant group, consisting of arrestin and binding protein Dishevelled.

25. The analysis system according to claim 19, where the protease is selected from the group consisting of: protease nuclear inclusion virus engraving tobacco A (TEV), enterokinase, protease factor XA, thrombin, a protease PureAct™, protease Clean Cut™, protease PreScission™, serine/ser / thr protease, thiol protease, aspartate proteases, metalloproteases, amino peptidases, dipeptidase, tripeptides, carboxypeptidase and paticipated.

26. The analysis system according to claim 19, where a protein which activates a reporter, is self-activating.

27. The analysis system according to claim 19, where a protein which activates a reporter is a reporter.

28. The analysis system according to claim 19, where the protein that activates the reporter is an enzyme.

29. The analysis system according to claim 19, where a protein which activates a reporter, is a protein that causes a change in fluorescence of the reporter.

30. The analysis system according to claim 19, where the fluorescent protein is a reporter.

31. The analysis system according to claim 19, where a protein which activates a reporter selected from the group consisting of luciferase, Gaussia luciferase, Renilla luciferase, a fluorescent protein, green fluorescent protein, protein DsRed, peroxidase, β-galactosidase, β-lactamase.

32. The analysis system according to claim 19, where the first protein forms a fused protein with a protein which activates a reporter.

33. With the system analysis p.19, where the reporter is selected from the group consisting of: substrate galactosidase, peroxidase substrate, the substrate of luciferase and luciferin.

34. The analysis system according to claim 19, where the signal is selected from the group consisting of luminescence and color change.