KR20090075800A - A method of detecting and/or quantifying expression of a target protein candidate in a cell, and a method of identifying a target protein of a small molecule modulator - Google Patents

Abstract

A method for detecting and/or qualitating the expression of target protein candidate in a cell is provided to identify the target protein of small molecule modulator through expression of cDNA or the suppression of expression. A method for detecting and/or qualitating the expression of target protein candidate comprises: a step of introducing a first nucleic acid coding a marker protein; a step of introducing a second nucleic acid coding the target protein candidate; a step of inducing a vector in a cell; a step of detecting and/or qualitating the expression of marker protein; and a step of detecting and/or qualitating the expression of target protein candidate.

Description

METHODS OF DETECTING AND / OR QUANTIFYING EXPRESSION OF A TARGET PROTEIN CANDIDATE IN A CELL, AND A METHOD OF IDENTIFYING A TARGET PROTEIN OF A SMALL MOLECULE MODULATOR}

The present invention relates to methods for detecting and / or quantifying expression of target protein candidates in cells and for identifying target proteins of small molecule modulators.

The molecular basis of numerous aspects of cellular and protein function, particularly mammalian cell and protein function, remains undefined in most cases and, in the context of the visual screen of protein function at the cellular level, may enable the exploitation of genomic means. I need a way. This is advantageous for the identification of protein networks operating in and around the cellular biological system of interest and the target (s) of small molecule modulators identified by chemical genetics (Eggert and Mitchison, 2006) and similar high-speed screening approaches. . The current bottleneck in cell-level large screens of protein function is the identification of these targets (Peterson et al., 2006).

Moreover, in many cases, it is even more difficult to reliably and quantitatively detect the expression of target proteins in cells. Some approaches in the prior art have focused on the use of nucleic acid arrays and the effect on the effect of foreign material or disease state on the expression profile of nucleic acids in the array. This approach, in many cases, was at best qualitative. Moreover, the results of the nucleic acid array experiments yield large amounts of data that are mostly not directly applicable to further analysis. Moreover, this type of experiment only provides insight into the expression of nucleic acids, and any inferences related to protein expression are only indirect. Therefore, there is a need in the art for providing a method for detecting and / or quantifying intracellular protein expression that is easy to perform and analyze.

Small molecules are a useful tool for studying dynamic biological processes with high temporal resolution. Small molecules can also facilitate the discovery of therapeutic drugs. Pharmaceutically active small molecules can be found by screening chemical libraries for alterations of specific protein activity in pure proteins in in vitro assays or for the desired phenotype in cell level assays. In the past, cell level assays have generally been established to report the effect on activity of a single pathway or process, generally using luminescence or fluorescence signals as readout values measured in plate readers. In cell-imaging assays, cell fields are visualized using fluorescent tags and automated microscopy attached to different polymers. The data potentially contains large amounts of information related to phenotypic changes not only desired but also unexpected, and this approach is often referred to as "high content screening." The potential of the method in academia and industry for the discovery and characterization of useful compounds is large, but remains a challenge, particularly at the level of target identification and data analysis.

Small molecule imaging screens generally involve a number of steps. First, cells are generally plated in optical-bottom-multiwell plates, small molecules are processed and incubated for a suitable period of time. Subsequent combinations of small molecule fluorescent probes, immunofluorescence with antibodies and / or intracellular expression of fluorescent protein (eg, GFP) -tagged proteins cause the protein of interest to appear fluorescence. The latter method can be applied to both live cells and fixed cells. In a second step, the fluorescence image is captured by automated microscopy, where the image is analyzed to measure phenotypic changes to identify wells containing desired, unwanted or unexpected phenotypes. In small molecule screening methods, it is eventually necessary to identify the biochemical targets that caused the phenotypic change. This will obviously be difficult, especially if the phenotypic change is due to the shaking of one or more polymers.

As a result, there is a need in the prior art to provide an improved method for identifying target proteins of small molecule modulators that is easy to perform and applicable to assays. It is therefore an object of the present invention to provide a method that allows for the identification of targets, ie the identification of polymer targets for small molecule modulators, without complex biochemical assays. Furthermore, it is an object of the present invention to provide a method that can be used as a genome-wide broad approach for target identification, avoiding labor-intensive biochemical methods such as have been present in the prior art. By identifying a gene that modulates the activity of a small molecule modulator, a method is described that enables the identification of a polymer target for a small molecule modulator through the expression or inhibition of a group of potential target nucleic acids, preferably cDNAs. Specifically, the molecular targets of small molecules are identified through their ability to revert or to make them more powerful. The method enables target identification without complex biochemical assays, and thus is used as a genome-wide broad approach to target identification, unlike the labor-intensive biochemical methods that existed in the prior art (Peterson et al., 2006).

The object of the present invention is achieved by a method of detecting and / or quantifying the expression of a target protein candidate in a cell, comprising the following steps:

Introducing a first nucleic acid encoding a marker protein into the vector,

Introducing a second nucleic acid encoding said target protein candidate to detect and / or quantify expression in said vector such that said first and second nucleic acids are operably linked such that expression of said marker protein To be an indication of candidate expression,

Introducing the vector into a cell,

Detecting and / or quantifying the expression of said marker protein,

Combining the expression of the marker protein with the expression of the target protein candidate to detect and / or quantify the expression of the target protein candidate.

In one embodiment, the first and second nucleic acids are operably linked to the vector by one of the following arrangements:

a) the first nucleic acid is under the control of a first promoter, the second nucleic acid is under the control of a second promoter located separately from the first promoter, and the first and second promoters are identical in sequence Or have the same activity,

b) the first nucleic acid is under the control of a first promoter, the second nucleic acid is under the control of a second promoter located separately from the first promoter, and the first and second promoters are not identical in sequence , The activity of each of the promoters is predictable,

c) the first and second nucleic acids are under the control of a single promoter, and the first and second nucleic acids are spaced apart from each other by an interval of nucleotides comprising an internal ribosome entry site (IRES).

In one embodiment, the marker protein comprises a fluorescent protein, an antibody fragment, an epitope, an enzyme, a monomeric avidin, a peptide biotin mimetic, a chemical fluorophore or similar construct that produces a signal that is detectable through direct binding or optically. Peptides are detectable by peptides detectable through chemical bonds or reactions with organic molecules comprising.

In one embodiment, the IRES is an IRES from a virus, an IRES from a cell's mRNA, in particular picornaviruses such as polio, EMCV and FMDV, flaviviruses such as hepatitis C virus (HCV), pestiviruses such as From porcine cholera virus (CSFV), retroviruses such as murine leukemia virus (MLV), lentiviruses such as monkey immunodeficiency virus (SIV), and insect RNA viruses such as cricket paralysis virus (CRPV) IRES, and mRNAs of cells, for example translation initiation factors such as eIF4G and DAP5, transcription factors such as c-Myc and NF-κB-inhibitory factor (NRF), growth factors such as vascular endothelial growth factor (VEGF), Fibroblast growth factor 2 (FGF-2), platelet origin growth factor B (PDGF-B), homeogene genes such as antennapedia, surviving proteins such as apoptosis X-associated inhibition (XIAP), the IRES of the mRNA, e.g., BiP Origin and Apaf-1, and other cells.

In one embodiment,

If said first and second promoters are identical in sequence, they are selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters,

When the first and second promoters have the same activity, each of them is independently selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters,

If said first and second promoters are not identical in sequence and the activity of said promoter is predictable, each of said promoters is independently selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters,

The single promoter is selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters and the IRES is selected from the group comprising nucleic acids having a sequence selected from the group comprising SEQ ID NOs: 1-15 . In a preferred embodiment, the single promoter is a CMV promoter and the IRES is from EMCV. In a particularly preferred embodiment, said promoter is a CMV immediate early (IE) promoter (pCMV _IE ) and the IRES is from EMCV; Preferably said IRES from EMCV is a nucleic acid having a sequence selected from SEQ ID NOs: 9, 14 and 15, preferably 14 and 15, most preferably 14.

In one embodiment, the introduction of the vector into the cell is performed via transformation, transfection, electroporation, viral transduction, transduction, ballistic delivery.

Preferably, the detection and / or quantification is any optical detection method capable of quantitative measurement, in particular optical detection with spatial resolution, microscopy, fluorescence activated cell sorting, UV-visible spectroscopy, fluorescence or phosphorescence measurements, biometrics Luminescence measurements, more preferably the microscopy is performed by optical microscopy, brightfield microscopy, polarization microscopy, fluorescence microscopy, in particular confocal fluorescence microscopy, surface attenuation wave irradiation microscopy, fluorescence correlation spectroscopy, fluorescence Life span microscopy, fluorescence cross-correlation microscopy, post-bleach fluorescence recovery microscopy, line scanning imaging, point scanning imaging, structured illumination, deconvolution microscopy, photon count imaging.

In one embodiment, the target protein candidate and the marker protein are expressed as separate proteins in the cell.

The object of the present invention is also achieved by a method of identifying a target protein of a small molecule modulator, comprising the following steps:

Providing a first cell of a type capable of generating a signal when exposed to a small molecule modulator, said signal being a spatially degradable, optionally quantifiable signal, preferably by microscopy,

Exposing and spatially digesting said first cell with a small molecule modulator, optionally quantifying a first signal produced by said first cell as a response to said small molecule modulator,

Providing a second cell of the same type as said first cell, and

Performing the method of any one of claims 1 to 9 on said second cell,

While performing the method of any one of claims 1 to 9 on the second cell, after introducing the vector into the second cell, the second cell is exposed to the small molecule modulator and spatially Quantifying the second signal generated by the second cell in response to the small molecule modulator,

Comparing the first signal with the second signal and if there is a difference between the first signal and the second signal, the difference is due to the expression of the target protein candidate in the second cell, thereby reducing the Identifying said target protein candidate as a target protein of a molecular modulator.

Preferably, the first signal and the second signal can be detected by microscopy, spatially resolved and optionally quantified, where more preferably the first signal and the second The signal is a fluorescent signal.

In one embodiment, expression of the marker protein produces a third signal that can be spatially resolved and distinguished from the first and second signals, wherein preferably the third signal is detected microscopically. And spatially resolved and optionally quantified.

In a preferred embodiment, the third signal is a fluorescence signal, the first and second signals are fluorescent signals, and the third signal is distinguished from the first and second signals in the spectrum.

In one embodiment, the first signal and the second signal can be distinguished from each other only because of their respective amounts.

Preferably, the method according to the invention is carried out with more than one, preferably several target protein candidates, for a given small molecule modulator, more preferably for a given small molecule modulator, all Possible target protein candidates are performed.

In one embodiment, the method according to the invention is carried out using more than one, preferably several small molecular modulators.

The object of the present invention is achieved by a method of identifying a target protein of a small molecule modulator, comprising the following steps:

Providing a first cell of a type capable of generating a signal upon exposure of the cell to a small molecule modulator, said signal being spatially resolved, optionally quantified, preferably by microscopy ,

Exposing and spatially digesting the first cell with a small molecule modulator and optionally quantifying a first signal produced by the first cell as a response to the small molecule modulator, wherein the step comprises the target protein At a number of different concentrations of the small molecule modulator to determine the concentration (AC ₅₀ ) when half the first maximum activity of the small molecule modulator, which is half the maximum activity in the absence of inhibition of the candidate's expression,

Determining a concentration when half of the first maximum activity,

Providing a second cell of the same type as said first cell, and

Introducing into said second cell a small inhibitory RNA (siRNA) selected to inhibit the expression of a target protein candidate in said second cell, preferably said second cell in said second cell Transfecting said second cell with a small inhibitory RNA selected to inhibit expression of a target protein candidate,

Exposing and spatially digesting the second cell to the small molecule modulator, optionally quantifying a second signal produced by the second cell in response to the small molecule modulator, wherein the step is the target At a number of different concentrations of the small molecule modulator to determine the concentration (AC ₅₀ ) at half the second maximum activity of the small molecule modulator that is half the maximum activity in the presence of inhibition of expression of the protein candidate,

Determining a concentration when half of the second maximum activity,

Comparing the concentration at half the first maximum activity with the concentration at half the second maximum activity, and at half the first maximum activity at half the concentration and half the second maximum activity If there is a difference in concentration, the difference is due to inhibition of expression of the target protein candidate, thereby identifying the target protein candidate as the target protein of the small molecule modulator.

Preferably, the concentration when the half of the first and second maximum activities is half the maximum inhibition (IC ₅₀ ) and the small molecule modulator is the inhibitor.

In another embodiment, the concentration when half of the first and second maximum activities is at a concentration (EC ₅₀ ) when half of the maximum enhancement, and the small molecule modulator is an enhancer.

As used herein, it should be noted that the term “concentration at half the maximum activity (AC ₅₀ )” may refer to another ratio of activity in some embodiments. For example, "percentage at 90% of maximum activity (AC ₉₀ )" or another percently discriminated value such as 60%, 70% or 80% (AC ₆₀ , AC ₇₀ , AC _80, etc.) May be referred to. Again, in such a case, the percentage is subsequently applied to the terms "ICx" and "ECx", where "x" represents the above mentioned percentage of maximum active concentration, and "IC" in this case is a small molecule modulator inhibitor. "EC" means that the small molecule modulator is an enhancer. In a preferred embodiment, the "concentration when half of the maximum activity" is the concentration when the activity is at least 50% of the maximum value, ie "AC _{≧ 50} ".

Gene silencing is well known to those skilled in the art and can be accomplished in a number of ways (as outlined in Echeverri & Perrimon Nature, Reviews Genetics 2006).

Preferably, the first signal and the second signal are optical signals that are microscopically detected, spatially resolved, and optionally quantifiable.

More preferably, the first signal and the second signal are fluorescent signals.

The inventors have sought to devise a method that enables the identification of polymer targets for small molecule modulators through the expression of a group of potential target cDNAs or through their inhibition by identifying genes that regulate the activity of the small molecule modulators. . Specifically, the molecular targets of small molecules are identified through their ability to restore or potentiate the small molecule phenotype. The method allows for the identification of targets without the need for complex biochemical assays, thereby providing a genome-wide broad range of target identifications in contrast to the labor-intensive biochemical methods that existed in the prior art (Peterson et al., 2006). It is used as an approach.

In particular, keeping the first and second nucleic acids under the control of a single promoter and keeping the first and second nucleic acids apart from each other by an interval of nucleotides comprising an internal ribosome entry site (IRES). In one embodiment, the inventors of the present invention have sought to devise a system that is particularly useful for high density screening, since the expression level of the first nucleic acid matches the expression level of the second nucleic acid. The term "expression level of nucleic acid" as used herein is meant to refer to the expression level of mRNA or protein corresponding to the nucleic acid. In said "IRES-embodiment", said first and said second nucleic acids are in a 1: 1 relationship between expression and expression levels between each other. In contrast, other approaches involving the use of two different promoters can be associated with various problems. For example, promoter interference phenomena can occur and cannot be guaranteed for reliable coexpression of two nucleic acids. Thus, the "dual promoter approach", when used in a method for identifying a target protein of a small molecule modulator, is included in the present invention, but includes two nucleic acids under the control of a single promoter and containing the internal ribosome entry site. It is less desirable than the approach of keeping them apart from each other by spacing. For example, in a dual promoter approach, due to the presence of the resistant gene on the transcription unit away from the transcription unit expressing the gene of interest, it is not possible to ensure that the gene of interest is really expressed by the resistant cell. For example, if the gene of interest has an inhibitory effect on cell proliferation, or if it has a cytotoxic effect, the selection pressure on a separate transcription unit cannot resist non-selection on the gene of interest, thus resisting it. This leads to resistance clones expressing only the gene. The "IRES-approach" has no such problem, and consequently this is one of the advantages of this particular embodiment.

Moreover, in another aspect of the present invention involving siRNA, the inventors of the present invention can measure changes in concentration (AC ₅₀ ) when half of the maximum activity in target gene silencing. We have devised a method for identifying target proteins for small molecule modulators. In all the prior art methods of high density screening or high throughput screening known to the inventors, the above change in AC ₅₀ has not been measured, while only a single point of concentration, if any. When the single time concentration measurement is performed at high concentration levels, this generally results in a high probability of false positives, ie the method is not specific for all targets. Likewise, some small molecule modulators can be widely used above their "suitable" AC ₅₀ -concentrations, where toxic effects can be observed, and target detection is impossible. Moreover, in such a single concentration measurement, often the concentration effectively used is so low that no effect can be detected and thus the target cannot be identified. The inventors of the present invention devised a method of combining an AC ₅₀ titration experiment with a changeable AC ₅₀ and an appropriate change in the phenotype. Only appropriate targets varied the AC _{50, which} would likely be a target in single point concentration measurements, distinguishing it from the rest of the wrong genes. Thus, by performing AC ₅₀ -measurement in a method for identifying a target protein of a small molecule modulator using siRNA, the inventors of the present invention have devised a particularly reliable and highly integrated screening method.

As used herein, “target protein candidate” is meant to refer to any protein whose expression is to be detected and / or quantified. Preferably, the protein is subsequently suspected of targeting the action of small molecular compounds. The terms "small molecular compound" and "small molecular modulator" are used synonymously. As used herein, the term “target protein candidate” is suspected to be a target for small molecule modulators, but the doubt has not yet been established. If the "target protein candidate" is identified as the actual target for the small molecule modulator, the "target protein candidate" is the "target protein" of the small molecule modulator investigated.

As used herein, the term “operably linked” means that in an arrangement between two coding nucleic acids, an arrangement in which the expression of each protein encoded by its nucleic acid is in proportion to each other. . Thus, it is possible to detect and / or quantify the expression of a given protein encoded by a given nucleic acid to infer the presence and / or quantification of the expression of a second protein encoded by another nucleic acid. In its simplest form, the proportional relationship may be a linear relationship, but the present invention is not necessarily limited thereto.

As used herein, “internal ribosomal entry site” or “IRES” is meant to refer to some spacing of nucleotides within a coding sequence that causes translation to begin within the coding sequence at the level of mRNA. The protein resulting from the IRES-initiated translation is different from the protein in which it is located in its mRNA sequence. Generally, in eukaryotic cells, translation is initiated only at the 5'-end of the mRNA molecule, as 5 'cap recognition is required for assembly of the initiating complex. At present, it is believed that the IRES site mimics the 5 'cap structure. The IRES sequence was first discovered in RNA viruses but subsequently identified in a number of organisms. The IRES site is located in the 5 'untranslated region of the RNA and enables translation of the RNA in a cap independent manner. A number of IRES sites have been identified and are characterized by the above mentioned ability to initiate translation within mRNA in a cap independent manner. IRES sites have been comprehensively outlined (Martinez-Salas et al., Journal of General Virology, 2001, Vol. 82, 973-984, and Jackson, RJ, Translational Control of Gene Expression, 2000, Sonenberg et al., Pp. 127 -184, Cold Spring Harbor NY). In addition, a regularly updated database of identified IRES sites is available at http://www.iresite.org.

Particularly preferred IRES sites for use in the method according to the invention are IRES derived from viral RNA such as picornaviruses, flaviviruses, pestiviruses, retroviruses, lentiviruses and insect RNA viruses and translation initiation factors, transcription factors And IRES sites derived from cellular mRNA such as growth factors, homeogenes and surviving proteins. Particularly preferred IRES sites are IRES sites derived from picornaviruses and flaviviruses such as EMCV and HCV. Preferred IRES sites for use in the present invention include Drosophila melanogaster antennapedia, homeogene gene antennapedia and Ultrabitorax, human c-myc oncogene, human v-myc myelomyosis virus Related Oncogenes, Human Myelin Transcription Factor 2 (MYT2), Human Apoptosis Protease Activation Factor 1 (Apaf-1), Human Coxsackievirus B2 Strain 20, Brain Myocarditis Virus (EMCV), Cricket Panic Virus, Bovine IRES site from viral diarrheal virus 1, Hog Cholera virus (swine cholera virus) and hepatitis GB virus B (GBV-B).

More specifically, IRES sites derived from EMCV are particularly preferred, wherein preferably the IRES derived from EMCV is selected from SEQ ID NOs: 9, 14 and 15, more preferably from 14 and 15, most preferably 14 It is a nucleic acid having a sequence.

One idea underlying the present invention is the arrangement of the IRES-site as a bi-cistronic mRNA between two nucleic acids encoding two proteins in a eukaryotic mRNA molecule. In such cases, such IRES sites can drive translation of downstream protein coding regions independently of the 5'-cap structure bound to the 5'-end of the mRNA molecule. In this setup, both proteins are produced in the cell, and their expression is singly coupled. The first protein located in the first cistron is synthesized by a cap-dependent initiation approach, while the translation initiation of the second protein is located in the intersistron spacer region between the two protein coding regions. It is indicated by the IRES-site.

Methods of introducing nucleic acids into cells, such as transformation, transfection, electroporation, viral transduction, transduction and / or ballistic delivery, are known to those skilled in the art, for example outlined in the literature There are: Sambrook and Russell, 2006.

As used herein, “small molecule modulator” is meant to refer to a molecule that is not a protein and whose molecular weight does not exceed 10,000. In determining whether a molecule is a "small molecule modulator", it may be helpful to consider the so-called Lipinsky rule. Christopher Lipinsky has been working on the design of negative drugs and has formulated a set of five-law laws to rule out compounds that would not succeed due to poor absorption or permeability. Thus, according to Lipinski's law, if a molecule has the following, it will not work effectively as a small molecule drug or modulator:

1. more than 5 H-linked donors (generally the sum of NH and OH),

2. more than 10 H-bond recipients (generally the sum of N and O),

3. molecular weight (MW)> 500,

4. calculated log P>, and

5. Weak Inhibition (<100 nM) (Lipinsky et al., 1997, Experimental and computational approaches for estimating solubility and permeability in drug development and development settings. ADV. Drug Delivery REV.23 (1-3). And partition coefficients of molecules between hydrophilic environments. However, starting from this Riffinsky's law, the “small molecular modulator” as used herein may also have a molecular weight that exceeds 500 but does not exceed 10,000. More specifically, as used herein, “small molecule modulator” may also refer to oligopeptides or oligonucleotides whose molecular weight does not exceed 10,000.

Small molecules are also referred to herein as "modulators" when such small molecules affect the activity and / or cellular metabolism and / or phenotype of the protein. Experimentally, small molecule modulators of proteins are often identified using high throughput screening methods applied to chemical libraries. The library may be developed in solid or liquid phase and may consist of natural compounds and derivatives or synthetic molecules thereof (Hall et al., 2001, J. Comb. Chem., 3: 125-150). Chemical libraries are often formed using combinatorial chemistry, which sequentially alters the functional groups and molecular backbone of both precursor compounds (Burke et al., 2003, Science, 302: 613-618; Schreiber, 2000, Science , 287: 1964-1969). High throughput screening has been shown to be particularly useful in the area of drug development, pesticides and food screening.

A frequently used method of carrying out the method according to the invention is microscopy. There are many types of microscopy and are known to those skilled in the art. The term "microscopy" includes, but is not limited to, optical microscopy, confocal microscopy, and automated microscopy screening methods. Particularly preferred forms of microscopy are fluorescence microscopy, in particular confocal fluorescence microscopy, optical microscopy, fluorescence lifetime microscopy, fluorescence cross-correlation microscopy and polarization microscopy. The limitation of the microscope is determined by its resolution, which is determined by the resolution. In general, the resolution limit is 300 nm to 10 μm, preferably 300 nm to 1 μm, depending on the particular type of microscopy used. In this context, the term “spatially decomposing a signal” as used herein refers to a space at the limit as low as 300 nm to 10 μm, preferably 300 nm to 1 μm, which is generally defined as the Abbe limit. I mean the decomposition of my signal.

In this application, it is often referred to as "providing a first cell of type ..." and "providing a second cell of type ...". As will be apparent to one skilled in the art, in each of the above steps, one or more cells of a particular type will be provided, so that performing the method according to the invention is not limited to using only a single cell. In fact, the majority of experiments performed using the method according to the invention will be carried out in cell cultures of many cells of a particular type. Thus, "provide a first type of ... cell" and "provide a second type of ..." also provide "provide a first group of ... type cell" and "... of type cells Provide a second group ".

As used herein, the term "siRNA" is meant to refer to an interval of RNA that can be used to silence gene expression and / or interfere with gene expression. Thus, siRAN is also sometimes referred to as "small interfering RNA". The siRNA can be introduced into the cell in a number of ways, one of which is to transfect the siRNA into the cell. However, in another embodiment, the gene silencing can be achieved by first introducing it into a cell and allowing its expression therein, such that the short hairpin RNA is actually expressed in the cell by a suitable vector, for example a plasmid. Can be.

As used herein, the term "concentration when half the maximum activity" or "AC ₅₀ " means to refer to the concentration of the small molecule modulator when the small molecule modulator shows half the maximum activity. For example, the small molecule modulator may be an inhibitor, in which the activity is an inhibitor. As a result, the concentration at half the maximum active concentration of the small molecule inhibitor is "concentration at half the maximum inhibition" or "IC ₅₀ ". If the small molecule modulator is an enhancer, the concentration at half of the maximum activity is "concentration at half of the maximum enhancement" or "EC ₅₀ ".

Note that the term "concentration at half the maximum activity (AC ₅₀ )" as used herein may refer to another active fraction in some embodiments. For example, "concentration at 90% of maximum activity (AC ₉₀ )" or another percentage value as a distinguishing value, such as 60%, 70% or 80%, etc. (such as AC ₆₀ , AC ₇₀ or AC ₈₀ ) Can be used. Again, in this case, the percentage applies to the terms "ICx" and "ECx", where x represents the above mentioned percentage of maximum active concentration, "IC" is where the small molecule modulator is an inhibitor, and EC "is the case where the small molecule modulator is an enhancer. In a preferred embodiment, the "concentration when half of the maximum activity" is the concentration when there is at least 50% of the maximum activity, ie "AC _{≧ 50} ".

The inventors of the present invention have found that it is possible to identify target proteins of small molecule modulators by investigating the influence of protein expression in cell level assays, where the cell level assay is based on cell exposure to small molecule modulators. The signal is generated by the cell as a result of the exposure. The result of this cell level assay is determined depending on the degree of expression (or lack of expression) of the target protein candidate. If the expression or absence of expression of the target protein candidate has a detectable effect on the cell level assay results, the target protein candidate whose expression is artificially induced, increased or silenced is determined for the small molecule modulator used in the cell level assay. It is a target.

The drawings will be described below. here,

1 is a confocal laser scanning micrograph of a cell in which a green fluorescent protein and a red fluorescent protein are operably linked by an IRES site so that expression of either is used as a measure of expression of a second insert in the IRES vector. Shows; Panel A (top left corner) shows confocal images of GFP fluorescence in transfected cells, panel B (top right corner) shows confocal images of RFP fluorescence in the same cells, panel C (top right corner). Bottom left corner) represents the ratio image of the images in A and B representing any pseudochromatic scale with respect to the GFP: RFP ratio. The correlation between GFP and RFP intensity per pixel is shown in panel D (bottom right corner), along with the GFP intensity on the X axis graphed for RFP fluorescence. The correlation coefficient between the intensities in D is 0.92 +-0.023 (n = 5) as described herein,

FIG. 2 shows the effect of transfecting three IRES-vectors into endothelin A-receptor-GFP cell line and expressing (GFP = green fluorescent protein); "etar" is a stable cell line transfected with an endothelin A-receptor: IRES: RFP-vector (RFP = red fluorescent protein) and "etbr" is the same cell line, but endothelin B-receptor: IRES: RFP-vector "KOPr" was transfected with the same cell line, but with kappa-opioid-receptor: IRES: RFP-vector (FIG. 2A);

2B shows the same type of experiment, but this time using a kappa-opioid-receptor-GFP-cell line;

FIG. 3 shows the results of transfection of three IRES-vectors with different G-alpha-protein components, G-alpha s, G-alpha i and G-alpha q (Gs, Gi and Gq), respectively.

4 shows the use of an IRES approach for identifying target proteins;

4A shows the results of the effect of compound Go6976 on agonist induced internalization of endothelin A-receptors; (DMSO = dimethyl sulfoxide, ET-1 = endothelin-1);

FIG. 4B shows the results of transfection experiments of endothelin A-receptor-GFP-cells with IRES: RFP-build bearing protein kinase c-alpha or protein kinase c-beta; FIG.

FIG. 4C shows the same results as FIG. 4B but this time for several endosomes normalized to the control at each concentration (Go = Go6976);

5 shows quantification of NF-kappa-B nuclear-cytoplasmic transport in high throughput immunofluorescence assays;

FIG. 6 shows similar experimental results, where cells were transfected with specific or nonspecific scrambled siRNA and measured leptomycin sensitive NFkappa-B transport.

7 shows a schematic diagram of the constructs used in the experiments using the red fluorescent protein and the green fluorescent protein in Example 1,

8 shows an example of a commercially available vector that can be used for bicistronic expression of two protein inserts linked to an IRES site. For example, the commercial vector pIRES2-DsRed-Express-Vector available commercially from Clontech Laboratories, Inc., USA can be used. Alternatively, if a measurable / detectable protein, such as GFP or RFP, is inserted upstream of the IRES, the DsRed-Express-Gene, ie the red fluorescent protein, may be replaced with any gene of interest. There is a commercially available vector having multiple cloning sites upstream and downstream of the IRES site, such as the pIRES-vector from Clontech Laboratories, Inc., USA.

FIG. 9 shows the effect of siRNA on IC ₅₀ of leptomycin B on extranuclear outflow of NfκB when a suitable alternative siRNA is silenced under the same conditions as in FIG. 6 in terms of mechanism.

Moreover, the following sequences are described, wherein SEQ ID NO: 1 is NM_206445 Drosophila antennapia CG1028-RJ, transcriptional variant J (Antp), IRES from mRNA. IRES name: Antp-D (1323-1574, 252 bp), cited in the following literature: Oh SK, Scott MP, Sarnow P. (1992) homeogene gene antennapdia mRNA may initiate translational initiation by internal ribosomal binding 5'-noncoding sequences that confer. Genes. Dev. 6 (9): 1643-1653.

SEQ ID NO: 2 is IRES and is named: Antp-DE (1323-1730, 408 bp), which is cited in: Oh SK, Scott MP, Sarnow P. (1992) homeogenic gene antennapedia mRNA Comprises a 5'-noncoding sequence that confers translation initiation by internal ribosomal binding. Genes. Dev. 6 (9): 1643-1653, and Ye X., Fong P., Iizuka N., Choate D., Cavener DR (1997) Ultrabitolax and Antennapian 5 ′ untranslated regions induce an internal translational initiation that is regulated in development. Promote. Mol. Cell. Biol. 17 (3): 1714-1721,

SEQ ID NO: 3 is IRES and is named Antp-CDE (1-1730, 1730 bp), which is cited in the following literature: Oh SK, Scott MP, Sarnow P. (1992) 5′-noncoding sequences conferring translational initiation by internal ribosomal binding. Genes. Dev. 6 (9): 1643-1653, and Ye X., Fong P., Iizuka N., Choate D., Cavener DR (1997) Ultrabitolax and Antennapian 5 ′ untranslated regions induce an internal translational initiation that is regulated as it occurs. Promote. Mol. Cell. Biol. 17 (3): 1714-1721,

SEQ ID NO: 4 is an IRES from V00568 human mRNA encoding c-myc oncozin and is named: c-myc (1-395, 395 bp), cited in Stoneley M., Paulin FE, Le Quesne JP, Chappell SA, Willis AE (1998) The C-Myc 5 'untranslated region contains internal ribosomal entry fragments. Oncogene. 16 (3): 423-428,

SEQ ID NO: 5 is an IRES derived from NM_005378 human (Homo sapiens) v-myc myeloma myeloma virus-related oncogene, which is (algal) (MYCN) mRNA derived from neuroblastoma. IRES name: N-myc (989-1308, 319 bp), which is cited in Jopling CL, Willis AE (2001) N-myc translation is initiated through internal ribosomal entry fragments showing enhanced activity in neurons. do. Oncogene. 20 (21): 2664-2670,

SEQ ID NO: 6 is an IRES from AF006822 human Myelin Transcription Factor 2 (MYT2) mRNA and is full cds, IRES name: MYT2_997-1152 (997-1152, 156 bp), which is cited in the following reference: Kim JG, Armstrong R. C, Berndt JA, Kim NW, Hudson LD (1998) A secretory DNA-binding protein that is translated through the internal ribosomal entry site (IRES) and distributed in a discontinuous pattern in the central nervous system. Mol. Cell. Neurosci. 12 (3): 119-140,

SEQ ID NO: 7 is an IRES from AFO13263 human apoptosis protease activating factor 1 (Apaf-1) mRNA, is a complete cds, IRES name: Apaf-1 (345-577, 233 bp), which is cited in Coldwell MJ, Mitchell SA, Stoneley M., MacFarlane M., Willis AE (2000) Initiation of Apaf-1 translation by internal ribosomal entry. Oncogene. 19 (7): 899-905,

SEQ ID NO: 8 is an IRES from AY752946 human Coxsackievirus B3 strain 20 and is the entire genome, IRES name: CVB3 (1-750, 750 bp), which is cited in the following literature: Jang GM, Leong LE, Hoang LT, Wang PH, Gutman GA, Semler BL (2004) Structurally unique factors mediate internal ribosomal entry within the 5'-noncoding region of potassium channel mRNA, which is opened and closed by cell membrane voltage difference. J. Biol. Chem. 279 (46): 47419-47430, and Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) Internal ribosomal entry sites mediate translation of enhancer factor-1 of the lymphatic system. RNA. 11 (9): 1385-1399,

SEQ ID NO: 9 is an IRES from NC_OO1479 cerebrospinal myocarditis virus and is a whole genome, IRES name: EMCV (257-832, 576 bp), which is cited in Wang Z., Weaver M., Magnuson NS (2005) ) Unknown promoter activity in the DNA sequence corresponding to pim-1 5'-UTR. Nucleic Acids Res. 33 (7): 2248-2258,

SEQ ID NO: 10 is an IRES from NC_003924 cricket panic virus and is the entire genome, IRES name: CrPV_5NCR (1-708, 708 bp), cited in the following documents: Wilson JE, Powell MJ, Hoover SE, Samow P (2000) Naturally occurring dystronic cricket panic virus RNA is regulated by two ribosomal entry sites. Mol. Cell. Biol. 20 (14): 4990-4999,

SEQ ID NO: 11 is an IRES from NC_OO1461 bovine viral diarrheal virus 1 and is a full genome, IRES name: BVDV1_1-385 (1-385, 385bp), which is cited in the following literature: Chon SK, Perez DR, Donis RO (1998) Genetic analysis of internal ribosomal entry fragments of bovine viral diarrheal virus. Virology. 251 (2): 370-382,

SEQ ID NO: 12 is an IRES from Z46258 Hog cholera virus (pig cholera virus) 'Chinese' strain (C-strain; EP 0 351 901 B1), which encodes a polyprotein. IRES name: CSFV (1-373, 373 bp), which is cited in the following references: Rijnbrand R., Bredenbeek PJ, Haasnoot P. C, Kieft JS, Spaan WJ, Lemon SM (2001) Hepatitis C virus and other plastics Influence of downstream protein-coding sequences on internal ribosomal entry in nonviral RNA. RNA. 7 (4): 585-597,

SEQ ID NO: 13 is an IRES from NC_001655 Hepatitis GB Virus B, a full genome RES, IRES name: GBV-B (1023-1467, 445 bp), which is cited in Rijnbrand R., Abell G., Mutation Analysis of Lemon SM (2000) GB Virus B Internal Ribosome Entry Site. J. Virol. 74 (2): 773-783.

SEQ ID NO: 14 is an IRES from EMCV used in the Examples below.

SEQ ID NO: 15 is an IRES from EMCV published on the website www.iresite.org.

In addition, the following examples are provided to illustrate the invention, which do not limit the invention.

Example

Example 1

Cell-level Assay of Protein Function and Quantification of Protein Expression

Agonist-induced Receptor Internalization

Design a cellular level assay of protein function—eg, receptor internalization with agonist-induced G-protein coupled, and apply it so that the desired activity of the protein can be measured or visualized and measured in a highly integrated (visual) assay did. Typically, for agonist induced receptor internalization, this may include visual identification of cells in which the receptor is internalized as part of a cell population. This can be quantified, for example, by computer-assisted image recognition. The assay can be using a fusion protein for a receptor that can be expressed in a cell and can be visualized directly under a microscope, such as green fluorescent protein (GFP). It may also be an assay in which the endogenous cell receptor is visualized by indirect methods such as immuno-labeling or using fluorescent agonists. Assay cell lines are typically stably transfected. In one embodiment, the receptor assay is visualized using 488 nM excitation and a suitable luminescence filter.

Ideation suggests the following approach to test the effect of protein expression (target) in the assay. The target is expressed with transcription related reporters distant on the spectrum as used in the assay. It is designed to approximate a linear relationship between target expression and reporter expression. Methods for achieving this include using internal ribosomal entry sites, two identical active promoters that drive the target and the reporter, or two promoters capable of predicting activity. In the simplest case, the reporter's expression is a measure / expression measurer that measures the expression of the target. Here, the inventors of the present invention describe the use of internal ribosomal entry sites.

Experiments were performed as follows: Target :: Reporter constructs were cationic lipid: DNA complex trans, as described in standard protocols (eg, Sambrook and Russell, Molecular Cloning, 3rd Edition, Chapter 16, CSH press, 2001). Spectroscopy, calcium phosphate mediated transfection, polymer based transfection methods, dendrimers). These methods are not very efficient and some reporter expression is detected. This provides a group of cells that express the target at a measurable level, and the impact of expression can thus be determined by visualizing the assay in cells expressing the target at different levels. Accordingly, the general weakness of cell transfection is exploited by virtue of the cell-based resolution of microscopy that allows the identification of single cells. Thus, the assay is measured on untransfected cells and cells that are transfected to express the target at a range of concentrations in a single experimental image.

One copy of the green fluorescent protein was inserted into the internal ribosomal entry vector, so that the IRES and red fluorescent protein (dsRED expression-monomeric red fluorescent protein, later referred to as RFP) were located downstream thereof (FIG. 7). After transiently transfecting HEK293 cells with the vector whose expression is controlled by the pCMW promoter (see FIG. 8), the fluorescence intensities of the green and red fluorescent proteins were determined using excitation (488/532 nm). Confocal laser scanning microscopy with more than 25 sugar cells and detection optics adapted to quantify the expression of the two fluorophores separately were measured (FIG. 1). The correlation coefficient determined between GFP and RFP intensities in confocal images of transfected cells was 0.91 + -0.023 (n = 5), indicating that RFP or GFP expression was measured for expression of each other insert in the IRES vector. It can be used as.

A series of cDNAs is cloned into an IRES: RFP vector encoding a) G-protein coupled receptor, b) small G-protein or c) protein kinase, and the sequence of IRES used in the above and all subsequent experiments is EMCV. SEQ ID NO: 14, which is derived from IRES. Initially, IRES vectors were transiently expressed in cell lines stably expressing the fusion protein between the endothelin A receptor and the c-terminal copy of the green fluorescent protein. Transfection efficiency for the cell line was about 50% by transfection with IRES vectors and monitoring RFP expressing cells. The vectors include any of the cDNAs encoding endothelin B receptor, kappa opioid receptor or endothelin A receptor 5 'to IRES and RFP. The effect of expression of the heterologous / exogenous receptor on agonist induced internalization of the endothelin A receptor was then measured. Two hours after cell stimulation with 40 nM endothelin-1, cells were imaged using automated confocal microscopy. Subsequently, an image of GFP-labeled endothelin A receptor, which is stably expressed in the cell line, may be used to measure the fraction of cells in which receptor endocytosis occurs, and the RFP image is transfected with an IRES vector in the same image. It was used to measure the degree of inclusion of GFP-labeled endothelin A receptor in cells.

This was done using both single blind manual counting and automated image analysis using periodicals for users in commercial image analysis packages (Metamorph offline, Molecular Devices, CA). Cells are divided as controls (non-RFP-expressors) or expressers (RFP producers) and expresser cells are low, medium and high expressor categories based on integrated RFP intensity / pixel in the image. Divided by. The number of cells showing receptor internalization was automatically counted in these four categories and expressed as a fraction of the total number of cells in each category.

a) Three IRES vectors are transfected separately to the endothelin A receptor-GFP cell line: endothelin A receptor: IRES: RFP, endothelin B receptor: IRES: RFP, and kappa opioid receptor: IRES: RFP . Compared with control cells in the same image, expression of ETAR: IRES: RFP had minimal effect on ETAR-GFP internalization, while both endothelin B receptor and kappa opioid receptors significantly reduced ETAR-GFP internalization ( 2A). This suggests that protein expression itself is a viable screening method for proteins involved in one pathway.

b) To demonstrate that the method had selectivity regardless of the assay, it was repeated using a kappa-opioid receptor-GFP cell line, where the agonist induced opioid receptor for any receptor expressed using the IRES vector. No significant difference was measured in the assay for internalization (FIG. 2B).

To further measure the utility of expression, IRES: RFP vectors were constructed for the expression of three G-alpha protein members of the heterologous trimeric G-protein, Gαs, Gαi and Gαq. On expression in ETAR-GFP cell line, two G-proteins significantly reduced internalization (Gαs, Gαi), while Gq had no significant effect compared to control cells expressing RFP alone (FIG. 3A).

This demonstrates that expression analysis strategies are involved in receptors and small signaling proteins.

c) A series of vectors comprising protein kinase C isotype, protein kinase C alpha or protein kinase C beta inserted at 5 'of IRES: RFP was transfected into an endothelin-A-receptor-GFP fusion protein stable cell line. . As shown in FIG. 4, expression of the protein kinase C isotype is endothelin-A-receptor-fusion- as compared to cells transfected with an IRES: RFP vector that lacks a 5 ′ insert before the IRES and expresses only RFP. There was no significant change in agonist induced internalization of the protein.

Example 2

Identification of Target Proteins for Small Molecular Modulators

The inventors of the present invention determined that the IRES approach can also be used to identify target proteins for compounds.

Agonist-induced internalization of the endothelin A receptor is blocked in excess of 85% when cells are exposed to the compound go6976, a micromolar concentration of protein kinase C inhibitor (Martiny-Baron et al., 1993) (FIG. 4A).

Protein kinase C has a number of isotypes and protein ETAR-GFP cells to determine whether protein kinase C alpha or protein kinase C beta isotypes can be targeted to Go6976 in endothelin A receptor internalization assays. Transfection with IRES: RFP constructs carrying kinase C alpha or protein kinase C beta.

Fugene6, 표적 시스템 타게펙트 및 그의 변형물, 리포펙타민 등과 같은 시판되어 입수가능한 방법 및 프로토콜을 이용하는 리포펙틴. 이어서, 트랜스펙션된 세포와 트랜스펙션되지 않은 세포를 모두 포함하는 세포를 농도 범위를 높여가는 Go6976 에 노출시키고, 세포를 아고니스트 엔도텔린-1 으로 자 극했다. 엔도텔린 A 수용체 활성의 측정치인, 세포 당 엔도좀의 갯수는, RFP 을 발현하는 트랜스펙션된 세포의 집단에서 그의 대조군인, 트랜스펙션되지 않은 이웃하는 세포와 비교하며 반자동화 영상 분석으로 결정했다. Cells were transfected using standard methods (outlined in Sambrook and Russell, 2006). In particular, Roche

Lipofectin using commercially available methods and protocols such as Fugene6, target system targetfects and variants thereof, lipofectamine and the like. Subsequently, cells containing both transfected and non-transfected cells were exposed to Go6976 at increasing concentrations, and the cells were stimulated with agonist endothelin-1. The number of endosomes per cell, a measure of endothelin A receptor activity, was determined by semi-automated imaging analysis and compared with its control, non-transfected neighboring cells in a population of transfected cells expressing RFP. did.

The expression effect of protein kinase C alpha in PKCα: IRES: RFP transfected cells was compared to control untransfected cells in the same field of view on Go6976 over a range of experimental concentrations using automated confocal microscopy. Typically, cells are first exposed to Go6976 in a predetermined concentration range for 120 minutes in 1% serum medium, and then the medium contains the same concentration of Go6976, but supplemented with agonists (tenfold agonist EC50: 40 nM). Exchange with medium. After incubation, cells were imaged using an automated high throughput confocal microscope (eg Opera, manufactured by Evotec Technologies, Hamburg). Images defining the endothelin-A-receptor-GFP distribution were obtained as images of separate RFP expresser cells in the spectrum. Two color images per cell area were aligned to correct for optical vignetting and mechanical misalignment (at levels of XY displacement of 1-3 μm; Metamorph Offline, Molecular devices corporation). Non-RFP expresser cells are defined as controls in the image, RFP expresser cells are segmented and then divided into low, medium and high (lower 20%, medium, upper 20%) presenters based on average RFP intensity per cell. did.

As expected, as the concentration of Go6976 increased, there was a significant decrease in the number of endosomes per cell in control cells (FIG. 4B left). Similar reductions were observed in cells expressing PKCα: IRES: RFP (left in FIG. 4B), indicating that protein kinase C alpha did not reverse the effect of compound Go6976. In contrast, the expression effects of protein kinase C beta in PKCβ: IRES: RFP transfected cells were different. Increasing Go6976 concentration reduced the number of endosomes in control cells, but endosomes were produced even at maximum concentrations of Go6976 in PKCβ: IRES: RFP expressing cells (FIG. 4B). Accordingly, protein kinase C beta was identified as the target of Go6976 in this assay because it provides a 'gain of function' and reverses the phenotype and the effects of Go6976.

The identification of protein kinase C beta as a target of Go6976 is firmly demonstrated when normalizing the number of endosomes for the control at each concentration (FIG. 4C). As is evident in FIG. 4C, protein kinase C alpha expression and analysis using this system do not reverse the effect of Go6976, while protein kinase C beta expression reverses the phenotype and is therefore a target of Go6976 in the assay.

Example 3

Identification of Target Proteins for Small Molecular Modulators Using siRNAs

Nuclear Transport of Transcription Factor Nuclear Factor Kappa B

In addition to the above method comprising gene expression as a 'gain function' screening for drug targets, the inventors also find that RNA interfering is used to reduce protein expression, and then to the required of compounds to provide 50% inhibition in the assay. Changes in AC ₅₀ / IC ₅₀ concentrations explain how to determine if it is the target of the drug in question. This is a 'loss of function' screening.

For nuclear transport as the main evidence, the translocation of the transcription factor nuclear factor kappa B was measured using high throughput immunofluorescence detection of the implicitly expressed NF-κB complex. Briefly, HeLa cells were washed twice with PBS, then fixed with 4% (w / v) paraformaldehyde in PBS for 10 minutes and then with PBS. Permeation was performed for 10 minutes using 0.1% TX-100 PBS, cells were washed in PBS, and then overnight using a 1: 200 dilution of rabbit anti-NF-κB in 10% goat serum-PBS. Incubation was carried out at ℃. Plates were washed three times on an orbital rotator for 10 minutes using PBS. Incubate 1: 1000 Alexa-488 goat anti-rabbit secondary antibody with the cells for 60 minutes at room temperature and wash the cells three times for 10 minutes with PBS on an orbital shaker, followed by 5 μM DRAQ5 (DRAQ5 in PBS). = Nuclear dye) was added for 10 minutes at 37 ℃.

Leptomycin B was used to block nuclear transport and derived that the IC ₅₀ is 2 ng / mL or 4.4 nM (FIG. 5). Expression of the extranuclear efflux protein Exportin 1 / CRM1 was reduced by transfection of small inhibitory RNAs targeting the Exportin 1 sequence, and the effect of leptomycin B on NFκB transport was evaluated as above. Exportin 1 / CRM1 knockdown using siRNA was estimated to be 50% using GFP silencing in the GFP expressor stable cell line, which was also a criterion at the same time (not shown). IC ₅₀ for leptomycin B in Expotin 1 / CRM1 knockdown cells was significantly reduced by more than 10-fold compared to control cells transfected with scrambled siRNAs with no known homology to human genes (FIG. 6). Thus, CRM1 / Expotin1 has been identified as a target of leptomycin B as predicted in the literature (Fornerod et al., 1997), and this method of targeting identification is valuable and has a role in screening at a broader range of genome levels. I confirmed that I would.

More specifically, FIG. 5 shows the quantification of NFκB nuclear-cytoplasmic transport by high throughput immuno-fluorescence assay. (a) Cytoplasmic NF-κB accumulates in the nuclei of HeLa cells treated with 0 (left panel), 1 ng / mL (center panel) or 20 ng / mL leptomycin B for 40 minutes, then fixed and anti- Detection was done using NFκB and Alexa 488 secondary antibodies, and nuclei stained using 10 μM Draq5. Scale bar 20 μm. (b) The distribution of NF-κB in the cytoplasm is indicated by the red being the nucleus and the green being the NF-κB position, which appears above the nucleus in a series of XZ (top row) and XY (center row) virtual images from left to right. Virtual nuclear position distribution image showing distribution. The bottom row shows the cell labeling status after treatment with green NF-κB labeled leptomycin B at 0, 1, 5, 10, 20 ng / mL in duplicate from the nuclear staining from left to right. Scale bar 5 μM. (c) Nuclear inflow quantification on the virtual image (top panel) and cell imaging (bottom panel). (d) Determination of EC ₅₀ for leptomycin based on innuclear location of NF-κB, incubation with 0-20 ng / mL leptomycin B, NF-κB detection and nuclear staining on microtiter plates. The EC ₅₀ set for leptomycin B was 2.4 ng / mL in the 95% confidence interval of 1.9 to 3 ng / mL with R ² as 0.9957 and is representative of more than five assays. All images were obtained on automated confocal.

6 shows quantification of NF-κB nuclear-cytoplasmic transport by high throughput immuno-fluorescence assay. Cells were transfected with crm1-specific or scrambled siRNAs, and leptomycin sensitive NFkB transport was measured after 24 hours. LMB curves were fitted using GraphPad Prism and R ² exceeded 0.95. All images were obtained on automated confocal (LMB = leptomycin B).

Example 4

FIG. 9 shows the effect of siRNA on IC ₅₀ of leptomycin B on nuclear influx of NFκB when silencing an alternative siRNA unrelated in terms of mechanism, under the same conditions as described in Example 3 above. As in Example 3, the translocation of the transcription factor nuclear factor kappa B was measured using high throughput immunofluorescence detection of the inherently expressed NF-kappa B complex. Briefly, HeLa cells were washed twice with PBS, then fixed for 10 minutes with 4% (w / v) paraformaldehyde in PBS and then washed with PBS. Permeation was performed for 10 minutes using 0.1% TX-100 PBS, and the cells were washed with PBS and then at 4 ° C. overnight with 1: 200 dilution of rabbit anti-NF-κB in 10% goat serum-PBS. Incubated. Plates were washed three times on an orbital rotator for 10 minutes with PBS. Incubate the 1: 1000 Alexa-488 goat anti-rabbit secondary antibody with the cells for 60 minutes at room temperature and wash the cells three times for 10 minutes on an orbital shaker with PBS, followed by 5 μM DRAQ5 in PBS (DRAQ5 = Nuclear stain) was added at 37 ° C. for 10 minutes.

Leptomycin B was used for nuclear transport blocking and IC ₅₀ was measured. In the silencing experiments, scrambled siRNAs or siRNAs targeting green fluorescent protein or protein kinase C isotypes were used. Silent expression was shown using control scrambled siRNA, green fluorescent protein or protein kinase C isotype.

The data in FIG. 9 demonstrates the specificity of achieving this experiment performance. Silencing of a series of other cDNAs does not provide a phenotype and does not affect IC ₅₀ . The experiment of FIG. 9 was performed under the same silencing conditions as described in Example 3.

references

Echeverrri C.J. And Perrimon, N. (2006) High Throughput RNAi in Cultured Cells: A Guide for Users. Nature reviews genetics 7, 374-384

Eggert, U.S. And Mitchison, T.J. (2006) Small molecule screening by image analysis. Curr Opin Chem Biol. 2006, 10, 1-6

Fornerod, M., Ohno, M., Yoshida, M. and Mattaj, I.W. (1997) CRM1 is an outflow receptor for leucine-rich extranuclear outflow signals. Cell, 90, 1051-1060.

Protein by Martiny-Baron, G., Kazanietz, MG, Mischak, H., Blumberg, PM, Kochs, G., Hug, H., Marme, D. and Schachtele, C. (1993) indolocarbazole Go6976 Selective Inhibition of Kinase C Isozyme. J Biol Chem, 268, 9194-9197.

Peterson, J. R., Lebensohn, A.M., Pelish, H.E. And Kirschner, M. W. (2006) Biochemical Inhibition of Small Molecule Inhibitors: A Strategy for Identifying Inhibitor Targets and Signaling Pathway Members. Chem Biol, 13, 443-452.

Sambrook and Russell, Molecular Cloning 3rd Edition, Chapter 16, 16, CSH press 2001

The features of the invention, the appended claims and / or the drawings disclosed herein will be material for implementing the invention in its various forms, either individually or in any combination.

<110> Institut Pasteur Korea        Emans, Neil   <120> A method of detecting and / or quantifying expression of a target        protein candidate in a cell, and a method of identifying a target        protein of a small molecule modulator. <130> I30926PCT <160> 15 <170> PatentIn version 3.3 <210> 1 <211> 252 <212> DNA <213> Drosophila melanogaster <400> 1 agtttcaaaa tcaaaattga ggaataccaa ctagaggata aggctactta aggatcaaaa 60 aacaccaagg agacgagatt ttctaccaaa tcgagagacg aggggcaggt taatttcgtc 120 atttttggcc aagacagcaa atagaggaac agcaaagcga aaatcatttt atacctcaca 180 caacaactac acactaacta agattaggct acgcaactgt acattgtact taagtgttca 240 aagtatattt ag 252 <210> 2 <211> 408 <212> DNA <213> Drosophila melanogaster <400> 2 agtttcaaaa tcaaaattga ggaataccaa ctagaggata aggctactta aggatcaaaa 60 aacaccaagg agacgagatt ttctaccaaa tcgagagacg aggggcaggt taatttcgtc 120 atttttggcc aagacagcaa atagaggaac agcaaagcga aaatcatttt atacctcaca 180 caacaactac acactaacta agattaggct acgcaactgt acattgtact taagtgttca 240 aagtatattt agtttacttt gtatataaga aaagtagcta aaagcacgcg gacagggagg 300 caggagcacc acagtcacta gccactaagc agagtcacag tcacgatcac gttcactcca 360 ggatcaggac tcggggcggg atcagcagac gctgaggaag ctgccacg 408 <210> 3 <211> 1730 <212> DNA <213> Drosophila melanogaster <400> 3 ttcagttgtg aatgaatgga cgtgccaaat agacgtgccg ccgccgctcg attcgcactt 60 tgctttcggt tttgccgtcg tttcacgcgt ttagttccgt tcggttcatt cccagttctt 120 aaataccgga cgtaaaaata cactctaacg gtcccgcgaa gaaaaagata aagacatctc 180 gtagaaatat taaaataaat tcctaaagtc gttggtttct cgttcacttt cgctgcctgc 240 tcaggacgag ggccacacca agaggcaaga gaaacaaaaa gagggaacat aggaacagga 300 accagataat agtgacataa gcgacccttt cgcaaatatt ttggcgcaaa atgagcgggc 360 gccaagtgcc gcgtggtgga gccgcctgaa aatgacatgg aaaattcgcc gaaaatcgcg 420 cgttttggca gcatcaatcc caaagcacaa aattaatttc tatcataatt tctgggtgca 480 acacggaccc ataattgaat cgaatatagg gcttatctga tagcccggca gcaacattga 540 actttccggc tgcaaaggag acgacaccga gatcgccaat tttcgttggg ctcgttctct 600 gggctccggc gataagaaat ccatgctgat aaggacagga ggacggtctg cggcaaattg 660 aattcgattc tgacctgtat gaaagccagc ggagatacgg atacctctgg gtttatgggt 720 agaaaacgca gagcgtcgcg ccaacatcga aattatttgc gtttgcatct tctcgtcctt 780 tcgtttatcg ttctgattgc catcgtggtg gcgcggtttc tattaatttt gcttctgtat 840 cgtttgcaaa atctcaaaag attcaaaaag ttcgtcatca gcagccgcaa cacaaaaacc 900 aacgagtgta aagccgagca tacaaatatc aataaaaaca taaacattta cccaatctca 960 atctcaaaac attcgcatcg tttccacaca aatatgctta gttcgcccaa attgtgattg 1020 tatatatata tttaacggca ttaaatacaa aagattaagc cctaaattaa gtgtaaatct 1080 tacaaaacgt ctacgttttt aaacaagaaa ttgtgatatt atatattaat cgggaaattc 1140 gaagtatgag aacaaaacgg tgtatatatg taagtgggcg atgaacatca atgaatattt 1200 tagctgagca aagtacacac gaatgaatat aaatatacat gaaaatatat tttgggcacc 1260 gacttttaca ccacaattat atatcgatag aaaagacacg aaaacaatca cagaaaacta 1320 agagtttcaa aatcaaaatt gaggaatacc aactagagga taaggctact taaggatcaa 1380 aaaacaccaa ggagacgaga ttttctacca aatcgagaga cgaggggcag gttaatttcg 1440 tcatttttgg ccaagacagc aaatagagga acagcaaagc gaaaatcatt ttatacctca 1500 cacaacaact acacactaac taagattagg ctacgcaact gtacattgta cttaagtgtt 1560 caaagtatat ttagtttact ttgtatataa gaaaagtagc taaaagcacg cggacaggga 1620 ggcaggagca ccacagtcac tagccactaa gcagagtcac agtcacgatc acgttcactc 1680 caggatcagg actcggggcg ggatcagcag acgctgagga agctgccacg 1730 <210> 4 <211> 395 <212> DNA <213> Homo sapiens <400> 4 aattccagcg agaggcagag ggagcgagcg ggcggccggc tagggtggaa gagccgggcg 60 agcagagctg cgctgcgggc gtcctgggaa gggagatccg gagcgaatag ggggcttcgc 120 ctctggccca gccctcccgc tgatccccca gccagcggtc cgcaaccctt gccgcatcca 180 cgaaactttg cccatagcag cgggcgggca ctttgcactg gaacttacaa cacccgagca 240 aggacgcgac tctcccgacg cggggaggct attctgccca tttggggaca cttccccgcc 300 gctgccagga cccgcttctc tgaaaggctc tccttgcagc tgcttagacg ctggattttt 360 ttcgggtagt ggaaaaccag cagcctcccg cgacc 395 <210> 5 <211> 317 <212> DNA <213> Homo sapiens <400> 5 gtctggacgc gctgggtgga tgcggggggc tcctgggaac tgtgttggag ccgagcaagc 60 gctagccagg cgcaagcgcg cacagactgt agccatccga ggacaccccc gcccccccgg 120 cccacccgga gacacccgcg cagaatcgcc tccggatccc ctgcagtcgg cgggagtgtt 180 ggaggtcggc gccggccccc gccttccgcg ccccccacgg gaaggaagca cccccggtat 240 taaaacgaac ggggcggaaa gaagccctca gtcgccggcc gggaggcgag ccgatgccga 300 gctgctccac gtccacc 317 <210> 6 <211> 156 <212> DNA <213> Homo sapiens <400> 6 cgcccgtaaa atgggaacgt agaactcgtc atgacacttg tggtaaaata acattattcg 60 cttgcaaaaa acgatgtact ttgacaattt aagacacaaa aaaacacaac ctcgacaatt 120 taggagttag agaggtcgtg ttttgagatt agtgag 156 <210> 7 <211> 233 <212> DNA <213> Homo sapiens <400> 7 cagagatcca ggggaggcgc ctgtgaggcc cggacctgcc ccggggcgaa gggtatgtgg 60 cgagacagag ccctgcaccc ctaattcccg gtggaaaact cctgttgccg tttccctcca 120 ccggcctgga gtctcccagt cttgtcccgg cagtgccgcc ctccccacta agacctaggc 180 gcaaaggctt ggctcatggt tgacagctca gagagagaaa gatctgaggg aag 233 <210> 8 <211> 750 <212> DNA <213> Coxsackievirus <400> 8 ttaaaacagc ctgtgggttg atcccaccca cagggcccat tgggcgctag cactctggta 60 tcacggtacc tttgtgcgcc tgttttatac cccctccccc aactgtaact tagaagtaac 120 acacaccgat caacagtcag cgtggcacac cagccacgtt ttgatcaagc acttctgtta 180 ccccggactg agtatcaata gactgctcac gcggttgaag gagaaagcgt tcgttatccg 240 gccaactact tcgaaaaacc tagtaacacc gtggaagttg cagagtgttt cgctcagcac 300 taccccagtg tagatcaggt cgatgagtca ccgcattccc cacgggcgac cgtggcggtg 360 gctgcgttgg cggcctgccc atggggaaac ccatgggacg ctctaataca gacatggtgc 420 gaagagtcta ttgagctagt tggtagtcct ccggcccctg aatgcggcta atcctaactg 480 cggagcacac accctcaagc cagagggcag tgtgtcgtaa cgggcaactc tgcagcggaa 540 ccgactactt tgggtgtccg tgtttcattt tattcctata ctggctgctt atggtgacaa 600 ttgagagatt gttaccatat agctattgga ttggccatcc ggtgaccaat agagctatta 660 tatatctctt tgttgggttt ataccactta gcttgaaaga ggttaaaaca ttacaattca 720 ttgttaagtt gaatacagca aaatgggagc 750 <210> 9 <211> 552 <212> DNA <213> Encephalomyocarditis virus <400> 9 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt 60 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt 120 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt 180 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct 240 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt 300 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt 360 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa 420 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta 480 gtcgaggtta aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa 540 acacgatgat aa 552 <210> 10 <211> 708 <212> DNA <213> Cricket paralysis virus <400> 10 tttaataagt gttgtgcaga ttaatctgca cagcactagc cacaaagttc agtggtccca 60 attatgggat ataaaactca atggtcgacg gagtgacctg gaaatgctcc ccgtgagaaa 120 ccttgtttaa acgcacttgg accttctccg gcctcagcgc caggcatagc tggatcgtca 180 atcccaagtt tggacttaga cttgctctat gttacaattg ggaggagcgt gacttaccat 240 tggtcgttga tgacgttaat tccaaacaaa aacaacaaca acaacttagt ttaaataatt 300 ccggtcttta agcatcaggg tagaccgatg aggaataaag aaccctataa gactgattcg 360 ataccaagag ctggtgtctg agggattacg actcgcgtgg tcggacagca catgttagtc 420 atgtattatg gtgtttccca acacgtatct ttgctctagt attgaacttt agtgatgagt 480 gtacttggca tgtacacctg attagttagc ccttgtggat acgccactgc cgctcaggta 540 cagtaggaca taccccaaat attgggttcc actcgtagag aagaaaatta gtggcgaaac 600 gtacggttta tgtcaaagga gaaggtgatt ttaattgcac ctgaagcaac cctaggtctt 660 ccctttgatg tgagctaata aggagtaacc gattcttaca atgtgatc 708 <210> 11 <211> 385 <212> DNA <213> Bovine viral diarrhea virus <400> 11 gtatacgaga actagataaa atactcgtat acatattgga caacagaaaa taactattag 60 gcctagggaa tgaatccctc tcagcgaagg ccgaaaggag gctagccatg cccttagtag 120 gactagcata atgagggggg tagcaacagt ggtgagttcg ttggatggct taagccctga 180 gtacagggta gtcgtcagtg gttcgacgcc tcggtataaa ggtctcgaga tgccacgtgg 240 acgagggcac gcccaaagca catcttaacc tgagcggggg tcgcccaggc aaaagcagat 300 cgaccaatct gttacgaata cagcctgata gggtgctgca gaggcccact gtattgctac 360 taaaaatctc tgctgtacat ggcac 385 <210> 12 <211> 373 <212> DNA <213> Hog cholera virus <400> 12 gtatacgagg ttagttcatt ctcgtataca cgattggaca aatcaaaatt ataatttggt 60 tcagggcctc cctccagcga cggccgaact gggctagcca tgcccatagt aggactagca 120 aaacggaggg actagccata gtggcgagct ccctgggtgg tctaagtcct gagtacagga 180 cagtcgtcag tagttcgacg tgagcagaag cccacctcga gatgctacgt ggacgagggc 240 atgccaagac acaccttaac cctagcgggg gtcgctaggg tgaaatcaca ccacgtgatg 300 ggagtacgac ctgatagggc gctgcagagg cccactatta ggctagtata aaaatctctg 360 ctgtacatgg cac 373 <210> 13 <211> 445 <212> DNA <213> Hepatitis GB virus B <400> 13 accacaaaca ctccagtttg ttacactccg ctaggaatgc tcctggagca ccccccctag 60 cagggcgtgg gggatttccc ctgcccgtct gcagaagggt ggagccaacc accttagtat 120 gtaggcggcg ggactcatga cgctcgcgtg atgacaagcg ccaagcttga cttggatggc 180 cctgatgggc gttcatgggt tcggtggtgg tggcgcttta ggcagcctcc acgcccacca 240 cctcccagat agagcggcgg cactgtaggg aagaccgggg accggtcact accaaggacg 300 cagacctctt tttgagtatc acgcctccgg aagtagttgg gcaagcccac ctatatgtgt 360 tgggatggtt ggggttagcc atccataccg tactgcctga tagggtcctt gcgaggggat 420 ctgggagtct cgtagaccgt agcac 445 <210> 14 <211> 585 <212> DNA <213> Encephalomyocarditis virus <400> 14 gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540 gggacgtggt tttcctttga aaaacacgat gataatatgg ccaca 585 <210> 15 <211> 577 <212> DNA <213> Encephalomyocarditis virus <400> 15 ccccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg 60 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc 120 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa 180 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag 240 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg 300 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg 360 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa 420 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg 480 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca 540 cggggacgtg gttttccttt gaaaaacacg atgataa 577  

Claims (24)

A method for detecting and / or quantifying expression of a target protein candidate in a cell, comprising the following steps: Introducing a first nucleic acid encoding a marker protein into the vector, Introducing a second nucleic acid encoding said target protein candidate to detect and / or quantify expression in said vector such that said first and second nucleic acids are operably linked such that expression of said marker protein To be an indication of candidate expression, Introducing the vector into a cell, Detecting and / or quantifying the expression of said marker protein, Combining the expression of the marker protein with the expression of the target protein candidate to detect and / or quantify the expression of the target protein candidate. The method of claim 1, wherein the first and second nucleic acids are operably linked to the vector by one of the following arrangements: a) the first nucleic acid is under the control of a first promoter, the second nucleic acid is under the control of a second promoter located separately from the first promoter, and the first and second promoters are identical in sequence Or have the same activity, b) the first nucleic acid is under the control of a first promoter, the second nucleic acid is under the control of a second promoter located separately from the first promoter, and the first and second promoters are not identical in sequence , The activity of each of the promoters is predictable, c) the first and second nucleic acids are under the control of a single promoter and the first and second nucleic acids are spaced apart from each other by an interval of an internal ribosome entry site (IRES). 3. The chemical fluorophore of claim 1, wherein the marker protein is a fluorescent protein, an antibody fragment, an epitope, an enzyme, an avidin, a peptidic biotin mimetic, a direct fluorophore or an optically detectable signal, or And a peptide detectable by a peptide detectable through a chemical bond or reaction with an organic molecule comprising a similar structure. The method according to claim 2 or 3, wherein the IRES is a virus-derived IRES, a cell-derived IRES, in particular picornaviruses such as polio, EMCV and FMDV, flaviviruses such as hepatitis C virus (HCV). , Pestiviruses such as porcine cholera virus (CSFV), retroviruses such as murine leukemia virus (MLV), lentiviruses such as monkey immunodeficiency virus (SIV), and insect RNA viruses such as cricket paralysis virus IRES from (CRPV)) and mRNAs of cells, for example translation initiation factors such as eIF4G and DAP5, transcription factors such as c-Myc and NF-κB-inhibitory factor (NRF), growth factors such as vascular endothelial Growth factor (VEGF), fibroblast growth factor 2 (FGF-2), platelet origin growth factor B (PDGF-B), homeogene genes such as antennapedia, surviving proteins such as apoptosis X-associated inhibitor (XIAP), and Apaf-1, and other cell mRNAs such as IRES from BiP. The method of claim 2, wherein the method is as follows: If said first and second promoters are identical in sequence, they are selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters, When said first and second promoters have the same activity, each of them is independently selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters, If the first and second promoters are not identical in sequence and the activity of the promoter is predictable, each of the promoters is independently selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters The single promoter is selected from the group comprising the CMV, EF1, SV40, human H1 and U6 promoters, and the IRES is selected from nucleic acids having a sequence selected from the group comprising SEQ ID NOs: 1-15. The method according to any one of claims 1 to 5, wherein the introduction of the vector into the cells is via transformation, transfection, electroporation, viral transduction, transduction, ballistic delivery. How is it done. The method according to any one of claims 1 to 6, wherein the detection and / or quantification is any optical detection method capable of quantitative measurement, in particular optical detection with spatial resolution, microscopy, fluorescence activated cell sorting, UV- Method performed by visible light spectroscopy, fluorescence or phosphorescence measurement, bioluminescence measurement. 8. The method of claim 7, wherein the microscopy comprises optical microscopy, wide field microscopy, polarization microscopy, fluorescence microscopy, in particular confocal fluorescence microscopy, surface attenuation wave irradiation microscopy, fluorescence correlation spectroscopy, fluorescence lifetime microscopy, fluorescence Cross-correlation microscopy, post-bleach fluorescence recovery microscopy, line scanning imaging, point scanning imaging, structured illumination, deconvolution microscopy, photon count imaging. The method of any one of claims 1 to 8, wherein the target protein candidate and the marker protein are expressed as separate proteins in the cell. A method of identifying a target protein of a small molecule modulator, comprising the following steps: Providing a first cell of a type capable of generating a signal when exposed to a small molecule modulator, said signal being spatially resolved, optionally quantifiable, preferably by microscopy Signal, Exposing and spatially digesting said first cell with a small molecule modulator, optionally quantifying a first signal produced by said first cell as a response to said small molecule modulator, Providing a second cell of the same type as said first cell, and Performing the method of any one of claims 1 to 9 on said second cell, While performing the method of any one of claims 1 to 9 on the second cell, after introducing the vector into the second cell, the second cell is exposed to the small molecule modulator and spatially Quantifying the second signal generated by the second cell in response to the small molecule modulator, -Comparing the first signal with the second signal, if there is a difference between the first signal and the second signal, because there is a difference in the expression of the target protein candidate in the second cell, thereby reducing the small Identifying said target protein candidate as a target protein of a molecular modulator. The method of claim 10, wherein the first signal and the second signal can be detected by microscopy, spatially resolved, and optionally quantified. 12. The method of claim 11, wherein said first signal and said second signal are fluorescent signals. 13. The method of any one of claims 10-12, wherein the expression of the marker protein produces a third signal that can be spatially degraded and distinguished from the first and second signals. The method of claim 13, wherein the third signal can be detected microscopically, spatially resolved, and optionally quantified. 15. The method of claim 14, wherein said third signal is a fluorescence signal, said first and second signals are fluorescent signals, and said third signal is distinguished from said first and second signals in a spectrum. 16. The method of any one of claims 11 to 15, wherein the first signal and the second signal can only be distinguished from each other due to their respective amounts. The method according to claim 10, wherein for a given small molecule modulator, it is carried out with more than one, preferably several target protein candidates. 18. The method of claim 17, wherein for a given small molecule modulator, it is performed using all possible target protein candidates of a given organism genome. 19. The process according to any one of claims 10 to 18, carried out using more than one, preferably several small molecule modulators. A method of identifying a target protein of a small molecule modulator, comprising the following steps: Providing a first cell of a type capable of producing a signal when exposed to a small molecule modulator, said signal preferably being spatially resolved, optionally quantified, by microscopy, Exposing and spatially digesting the first cell with a small molecule modulator, optionally quantifying a first signal produced by the first cell in response to the small molecule modulator, wherein the step comprises the target protein At a number of different concentrations of the small molecule modulator to determine the concentration (AC ₅₀ ) at half the first maximum activity of the small molecule modulator, which is half the maximum activity in the absence of inhibition of the candidate's expression, Determining a concentration when half of the first maximum activity, Providing a second cell identical to said first cell, and Introducing into the second cell a small inhibitory RNA (siRNA) selected to inhibit the expression of the target protein candidate in the second cell, preferably inhibiting the expression of the target protein candidate in the second cell Transfecting said second cell with a small inhibitory RNA (siRNA) selected to Exposing and spatially decomposing the second cell to the small molecule modulator, optionally quantifying a second signal produced by the second cell in response to the small molecule modulator, wherein the step is the target At a number of different concentrations of the small molecule modulator to determine the concentration (AC ₅₀ ) at half the second maximum activity of the small molecule modulator, which is the concentration at half the maximum activity in the presence of inhibition of expression of the protein candidate , Determining a concentration when half of the second maximum activity, Comparing the concentration at half the first maximum activity with the concentration at half the second maximum activity, and at half the first maximum activity at half the concentration and half the second maximum activity If the concentrations are different, the difference is due to inhibition of expression of the target protein candidate, thereby identifying the target protein candidate as the target protein of the small molecule modulator. 21. The method of claim 20, wherein the concentration when half of the first and second maximum activities is at a concentration (IC ₅₀ ) when half of the maximum inhibition and the small molecule modulator is an inhibitor. The method of claim 20, wherein the concentration when half of the first and second maximum activities is concentration (EC ₅₀ ) when half of the maximum enhancement and the small molecule modulator is an enhancer. 23. The method of any one of claims 20 to 22, wherein the first signal and the second signal are microscopically detectable, spatially resolved, and optionally quantifiable. The method of claim 23, wherein the first signal and the second signal are fluorescent signals.

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