JP2010071744A - Method and kit for screening compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a series of techniques for easily screening a compound specifically bonded to hydrophobic target protein such as membrane protein or the like. <P>SOLUTION: The method of screening the compound specifically bonded to hydrophobic target protein includes the step (1) for bringing fused protein of the target protein and chaperonin into contact with a compound library containing various compounds and bonding a desired compound to the target protein part of the fused protein, the step (2) for separating the aggregate of the fused protein and the compound from the compound library, and the step (3) for identifying the compound bonded to the fused protein. A kit for simply performing screening is also disclosed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a compound screening method and a screening kit. More specifically, the present invention relates to a method for screening a compound that specifically binds to a hydrophobic protein such as a membrane protein, and to perform the screening method easily. This relates to a screening kit. The present invention is useful for screening drug discovery seed compounds and the like.

  Membrane proteins present on cell membranes are membrane receptor proteins that play an important role in transmitting and controlling information outside the membrane, opening the channel portion in response to external environmental changes and ligand binding Channel proteins, transporters that support mass transfer between membranes, and membrane enzymes responsible for signal transmission near the membrane, all of which have important functions for cells. A substance that binds to a membrane receptor protein can be a candidate substance as an agonist-type drug or antagonist-type drug that controls an information signal. Therefore, analyzing the binding properties between the receptor protein and the test compound is extremely important in drug development. Among proteins present in the vicinity of the cell membrane, an integral membrane protein is strongly hydrophobic and different from a highly hydrophilic membrane surface protein on the membrane surface, and many proteins are embedded in the biological membrane. These hydrophobic proteins are potential target molecules for drug development.

  Screening for ligands that target a hydrophobic target protein is performed, for example, by evaluating the binding between a test compound that is a ligand candidate and a desired hydrophobic target protein, and selecting those that have specifically bound. . There are roughly two types of methods for evaluating binding, a method using living animal cells and an in vitro method using cell membrane fractions. The method using animal cells is a method of assaying using cells expressed by introducing a target protein gene of interest into animal cells such as CHO cells and HEK293 cells. On the other hand, as an in vitro method using a cell membrane fraction, there is a method using a labeled ligand as one example. For example, there is a method of screening using a cell membrane fraction prepared from an insect cell or animal cell in which a target protein is expressed on the membrane surface, using as an index the inhibition of binding of the labeled ligand and the target drug candidate compound to the target protein. However, these methods are effective methods for evaluating the characteristics of isolated compounds, but a great deal of labor is required in the process of selecting target drug candidate compounds from a large number of compound populations (compound libraries). Is required.

  An ALIS method (Automated Ligand Identification System) has been proposed as one method for isolating target drug discovery candidate compounds from a large number of compound populations (Non-patent Document 1). This method is effective when a highly hydrophilic protein is the target protein. Specifically, first, a target protein is contacted with a compound library containing thousands of compounds in a solution. The target protein to which the desired compound is bound is then separated from the compound library by HPLC. The separated target protein is treated with an organic solvent, and the bound compound is isolated from the target protein. Finally, the structure of the compound (ligand) isolated by LC / MS / MS is determined.

  However, in the ALIS method, it is necessary to concentrate and use the target protein at a high concentration of 10 mg / mL or more. Therefore, it is effective when the target protein is highly water-soluble, but is not suitable for highly hydrophobic proteins such as membrane proteins. In addition, since hydrophobic proteins such as membrane proteins are difficult to express in large quantities even using genetic recombination techniques, it is very difficult to apply the ALIS method, which requires a large amount of target protein, to hydrophobic proteins. Cost. Furthermore, in order to maintain a hydrophobic protein in a solubilized state at a high concentration while maintaining its higher order structure, appropriate treatment with a surfactant or the like is required, and a great deal of trial and error is required.

Kuroki et al., SAR News, Japan Pharmaceutical Association, October 1, 2005, No. 9, p. 2-

  In view of the above problems, an object of the present invention is to provide a series of techniques for easily screening for a compound that specifically binds to a hydrophobic target protein.

The invention according to claim 1 for solving the above-described problem is a method for screening a compound that specifically binds to a hydrophobic target protein, and includes the following steps (1) to (3):
(1) contacting the target protein and chaperonin fusion protein with a compound library containing a plurality of types of compounds, and binding a desired compound to the target protein portion of the fusion protein;
(2) separating the conjugate of the fusion protein and the compound from the compound library;
(3) identifying a compound bound to the fusion protein;
A method for screening a compound characterized by comprising

  Chaperonin is a kind of molecular chaperone and is a complex protein composed of subunits (chaperonin subunits) having a molecular weight of about 60,000. A typical chaperonin is a cylinder having a total molecular weight of about 800,000 to 1,000,000 having a two-layer structure in which two ring-shaped structures composed of 7 to 9 chaperonin subunits are non-covalently associated via a ring surface. A huge complex is formed. Chaperonins contain other proteins inside and can fold correctly. In the method for screening a compound of the present invention, a fusion protein of a target protein and a chaperonin is used instead of a single molecule target protein. Can exist in Therefore, it is possible to target a hydrophobic protein that has been difficult in the prior art. Furthermore, since the action of chaperonin is used, it is not necessary to use a surfactant or the like. Furthermore, in the present invention, each step is performed in a state where the target protein is basically stored in the ring structure of chaperonin, so that the target proteins can be prevented from approaching and aggregating. Therefore, the contact efficiency between the compound library and the target protein is high.

  The “fusion protein of target protein and chaperonin” in the present invention refers to “a fusion protein of target protein and chaperonin subunit” or “a fusion protein of target protein and chaperonin subunit conjugate”. Here, a chaperonin subunit linked body (sometimes referred to as a chaperonin linked body) refers to an artificial protein in which two or more chaperonin subunits are linked in series via peptide bonds. Details of the chaperonin subunit conjugate are described in, for example, WO 02/052029 pamphlet.

  The hydrophobic protein is a protein having a relatively high content of hydrophobic isoleucine, phenylalanine, leucine, valine, methionine, tryptophan, proline and alanine among the constituent amino acid residues. Hydrophobic proteins are generally difficult to dissolve in water (slightly soluble, sparingly water-soluble, insoluble) and difficult to express in recombinants (hardly expressible).

  A configuration in which the target protein is a membrane protein is recommended (Claim 2).

  In the step (2), a configuration in which the conjugate is separated by at least one method selected from chromatography, molecular sieving method, filter separation method, and precipitation method is recommended (Claim 3). In the step (3), a configuration for identifying a compound by mass spectrometry is also recommended (Claim 4).

  The invention according to claim 5 is the compound screening method according to any one of claims 1 to 4, wherein the fusion protein is a fusion protein of a target protein and a chaperonin subunit conjugate. .

  Chaperonin subunit conjugates have been shown to form ring-like structures similar to natural chaperonins, supplementing single molecule chaperonin subunits as needed (Furuya et al., Protein Science, 2005, 14 , 341). In the chaperonin subunit linked body, the subunits are strongly linked by peptide bonds, so that the formed ring-shaped structure is more stable. Therefore, according to the present invention employing the chaperonin subunit conjugate, the target protein is more reliably folded.

  The invention according to claim 6 is the compound screening method according to any one of claims 1 to 5, wherein the chaperonin forms a single layer structure.

  A chaperonin that forms a ring-shaped structure (single ring) composed of one layer is also known. In the present invention, a chaperonin that forms a single-layer structure is used, so that the target protein and the compound are easy to contact. Therefore, the efficiency of screening is improved.

  A seventh aspect of the present invention is a kit for use in the compound screening method according to any one of the first to sixth aspects, comprising the fusion protein and the compound library. This is a screening kit.

  The present invention relates to a compound screening kit, and includes the fusion protein and the compound library. According to the screening kit of the present invention, the screening method for the compound of the present invention can be easily performed.

  According to the compound screening method of the present invention, a compound that specifically binds to a hydrophobic target protein such as a membrane protein can be screened from a compound library with high efficiency.

  According to the screening kit of the present invention, a compound that specifically binds to a hydrophobic target protein such as a membrane protein can be easily screened.

The compound screening method of the present invention screens a compound that specifically binds to a hydrophobic target protein, and includes the following three steps.
(1) contacting the target protein and chaperonin fusion protein with a compound library containing a plurality of types of compounds, and binding a desired compound to the target protein portion of the fusion protein;
(2) separating the conjugate of the fusion protein and the compound from the compound library;
(3) identifying a compound bound to the fusion protein.

  The hydrophobic target protein is typically a membrane protein such as a transmembrane receptor protein, a membrane transport protein pump, a channel protein, or a membrane-bound enzyme, but is not limited thereto.

  In the present invention, a fusion protein of a target protein and chaperonin is used. In general, chaperonins are roughly classified into group 1 type and group 2 type. Chaperonins present in bacteria and eukaryotic organelles are classified as group 1 type, all of which form a ring structure in which seven chaperonin subunits having a molecular weight of 60 kDa are linked in a ring, and two rings form a two-layer structure. , Form a 14-mer homo-oligomer. An example of type 1 chaperonin is GroEL, which is a chaperonin derived from E. coli. Group 2 chaperonins, on the other hand, are found in eukaryotic cytoplasm and archaea, and are usually 16-18 mer homo- or hetero-oligomers in which rings consisting of 8-9 chaperonin subunits are connected in two layers. Forming. The chaperonin employed in the fusion protein used in the present invention may be either group 1 type or group 2 type.

  When the step (1) is performed in a solution, the higher the concentration of the fusion protein in the solution, the easier it is to separate the conjugate of the fusion protein and the compound from the compound library. Identification is also easy. On the other hand, when the concentration of the hydrophobic protein becomes high, there is a risk of forming an aggregated precipitate. In the present invention, since it is considered that the hydrophobic target protein is stored in the chaperonin ring in each step, aggregation of the target proteins can be suppressed and contact with the compound library at a high concentration can be achieved. The molecular weight of the target protein is preferably a molecular weight that can be stored in the ring structure of chaperonin, for example, 100,000 or less, more preferably 60,000 or less.

  Each process will be described sequentially. In step (1), the fusion protein is contacted with a compound library. For example, a solution containing a plurality of types of compounds and a solution containing a fusion protein may be mixed. When a desired compound (ligand) is contained in the compound library, the compound binds to a target protein portion in the fusion protein to form a conjugate between the fusion protein and the compound.

  Examples of the compound constituting the compound library include, but are not limited to, low molecular compounds. For example, high molecular compounds such as peptides, proteins, and nucleic acids can also be targeted for screening in the present invention.

  In step (2), the conjugate of the fusion protein and compound formed in step (1) is separated from the compound library. There is no limitation in particular as a method for isolate | separating, For example, the method normally used for the isolation | separation refinement | purification of protein is employable. In a preferred embodiment, various types of chromatography, molecular sieving methods, filter separation methods, and precipitation methods are used alone or in combination. In the case of chromatography, in addition to conventional methods such as ion exchange chromatography and hydrophobic chromatography, tags such as histidine tag, FLAG tag, and GST tag are previously bound to the fusion protein, and separation is performed using these tags. You can also. In the case of molecular sieving, for example, column method (gel filtration), electrophoresis, charcoal dextran method and the like can be used. In the case of the filter separation method, for example, ultrafiltration can be used. In the case of the precipitation method, for example, salting out such as ammonium sulfate precipitation, fractional precipitation with an organic solvent, acid precipitation, isoelectric point precipitation, or the like can be used.

  In step (3), a compound bound to the fusion protein is identified. For example, the combined product of the fusion protein and compound separated in step (2) is subjected to protein removal treatment, and the bound compound is isolated. This compound is identified by an appropriate analysis method. In a preferred embodiment, the compound is identified by mass spectrometry. In particular, according to the LC / MS / MS method combining liquid chromatography (LC) and mass spectrometry (MS), it is possible to identify with high accuracy. In this case, if the mass spectrum of each compound constituting the compound library is known, the compound bound to the target protein can be efficiently identified. On the other hand, when identifying a compound selected from a peptide library or a nucleic acid library, analysis of a base sequence using a sequencer or the like or analysis of an amino acid sequence is effective.

  In a preferred embodiment, a fusion protein of a target protein and a chaperonin subunit conjugate is used as the fusion protein. That is, one in which at least two chaperonin subunits in the fusion protein are linked via peptide bonds is used. As described above, it is known that a chaperonin subunit linked body also forms a ring-like structure in the same manner as natural chaperonin while supplementing a single molecule chaperonin subunit as necessary. In addition, the number of linkages (N) of the chaperonin subunit N-linked product is particularly preferably an optimal number determined by the origin of the chaperonin, and in the case of group 1 type chaperonin, seven, In some cases, 8 or 9 is preferred. If N is an optimum number of these, a ring-shaped structure can be formed only by a chaperonin subunit linked body, and it is not necessary to supplement the chaperonin subunit of a single molecule.

  In the fusion protein used in the present invention, as long as the ability to self-assemble into a ring structure is maintained, a mutant chaperonin such as an amino acid mutant can be used as well as a natural chaperonin.

  In a preferred embodiment, a chaperonin that forms a single layer structure is employed. For example, in the case of chaperonin GroEL (SEQ ID NO: 7) derived from Escherichia coli, four charged amino acids (R452, E461, S463, V464) in the equator domain strongly contribute to the binding between the rings based on crystal structure analysis. I know. Therefore, by replacing the 452nd amino acid residue (arginine) with glutamic acid and the 461st (glutamic acid), 463 (serine) and 464th (valine) amino acid residues with alanine, the interaction between rings is reduced, It is known to constitute a single ring (Weissman et al., Cell, 1995, 83, 577-587). In addition, chaperonin 60 derived from eukaryotic mitochondria is usually purified as a single ring and thus can be obtained without special genetic manipulation (Viitanen et al., J. Biol. Chem., 1992, 267, 695-698). By making the chaperonin ring into a single layer, the accessibility of the ligand to the hydrophobic protein stored in the chaperonin ring is improved, and the target compound can be efficiently recovered.

  The fusion protein used in the present invention can be produced by expressing a fusion gene in which a gene encoding a chaperonin conjugate and a gene encoding a hydrophobic target protein are linked. For example, the fusion gene may be incorporated into an expression vector, the recombinant vector is introduced into an appropriate host to produce a transformant, and the fusion gene is expressed in the transformant. The host used at this time is not particularly limited, but microorganisms such as bacteria are preferable from the viewpoint of low culture cost, short culture days, and easy culture operation, and Escherichia coli is particularly easy to handle. More preferred.

  A compound screening kit can be constructed by combining the above-described fusion protein and a compound library. The kit may contain other reagents such as standard substances, ligands, various buffers and the like. The following is a configuration example of the kit. As the fusion protein, for example, “a fusion protein of a human endothelin receptor (hETAR) and a GroEL 7-fold conjugate” (see Examples described later) can be employed.

[Example of kit configuration]
・ Fusion protein (lyophilized product): appropriate amount ・ Low molecular compound library: appropriate amount ・ BQ123 (hETAR antagonist): appropriate amount ・ Dilution buffer: appropriate amount

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(1) Construction of expression vector for fusing with E. coli-derived chaperonin GroEL seven-fold ligation Using the genomic DNA of E. coli K12 as a template, CPN-F1 (SEQ ID NO: 1) and CPN-R1 (SEQ ID NO: 2) are used as a primer pair. As a result, PCR was performed to amplify a DNA fragment containing the GroEL gene (SEQ ID NO: 3). A SpeI site and an XbaI site derived from the primer were introduced into the 5 ′ end and 3 ′ end of this DNA fragment, respectively. Seven of these DNA fragments were ligated in series to produce a GroEL 7-fold linked body gene ((GroEL) 7 gene). The (GroEL) 7 gene and other necessary sequences were introduced into the NcoI / HindIII site of the pTrc99A expression vector (Amersham Pharmacia) to construct the expression vector pTrc (GV) 7TC2H (FIG. 1). In FIG. 1, “P” is a trc promoter, “groEL” is a GroEL subunit gene, “(GroEL) 7” is a (GroEL) 7 gene, “PS” is a sequence encoding a peptide linker (peptide linker site), “ “CS” represents a cloning site, “Ek” represents an enterokinase translation site, “His” represents a sequence encoding a histidine tag (His peptide site), and “T” represents a terminator. That is, pTrc (GV) 7TC2H is downstream of the trc promoter (GroEL) 7 gene, peptide linker site (having a sequence encoding a thrombin cleavage recognition site), cloning site, enterokinase translation site, His peptide site, stop codon , And a terminator is arranged. Then, by inserting a gene encoding a hydrophobic target protein into the cloning site of pTrc (GV) 7TC2H, a fusion protein of the GroEL 7-fold conjugate and the hydrophobic target protein can be expressed.

(2) Preparation of Endothelin A Receptor Gene Fusion protein in which human endothelin A receptor (human ETAR) is employed as a hydrophobic target protein, and a 7-fold mutant GroEL subunit is linked to the N-terminal side of human ETAR Was prepared by the following procedure.

  A cDNA clone of human ETAR gene was obtained from OriGene (accession number NM_001957, SEQ ID NO: 4). Using this cDNA clone as a template, PCR was performed using a primer represented by hETaR-F1 (SEQ ID NO: 5) and hETaR-R1 (SEQ ID NO: 6) as a primer pair to amplify a DNA fragment containing the human ETAR gene. A BglII site (5 'end) and an XhoI site (3' end) derived from the primer were introduced at both ends of this amplified DNA fragment. This amplified DNA fragment was introduced into a pTrc (GV) 7TC2H vector previously digested with BglII and XhoI to prepare a pTrc (GV) 7TC2H-ETAR vector.

(3) Production of fusion protein The fusion protein expression vector constructed in (2) above was introduced into Escherichia coli BL21 (DE3) pRARE strain to obtain a transformant. A 2 liter Erlenmeyer flask was charged with 700 mL of 2 × YT medium (16 g / L yeast extract, 20 g / L bactotryptone, 6 g / L sodium chloride, 100 μg / mL ampicillin, pH 7.5), and 2 transformants were added. Inoculated with ~ 3 platinum ears. After culturing at 110 rpm for 40 hours at 25 ° C., the cells were collected by centrifugation (5000 rpm, 10 minutes). The obtained cells were suspended in 20 mL of PBS (pH 7.4) containing 1% protease inhibitor cocktail (Nacalai Tesque) and 10 mM imidazole, and stored frozen at −80 ° C.

  The frozen cell suspension was thawed, subjected to ultrasonic disruption, and then subjected to ultracentrifugation, and the supernatant fraction was collected. The supernatant was further loaded onto a nickel chelate column (5 mL), and the fusion protein was eluted with a linear gradient of 500 mM imidazole / PBS, and then the fraction containing the fusion protein was concentrated. Furthermore, 10% ammonium sulfate was added to the fraction containing the fusion protein, and the mixture was applied to 5 mL of HiTrap Butyl FF column (GE Healthcare) previously equilibrated with 10% ammonium sulfate / 10 mM sodium phosphate buffer. Elution was performed with 10 mM sodium phosphate buffer, and the fraction containing the fusion protein was collected. This fraction was further subjected to gel filtration using TSKgel G4000SWXL pre-equilibrated with 50 mM HEPES-Tris (pH 7.4) buffer to purify the target fusion protein. The purity of the obtained fusion protein was about 95%.

(4) Screening of hit compounds from compound library Approximately 100 kinds of low molecular compounds were dissolved in dimethyl sulfoxide so as to be 400 μM each. Further, BQ123 (model hit compound), which is an antagonist of endothelin A receptor, was added and mixed so as to be 400 μM to obtain a compound library. This compound library was diluted 20 times with 100 mM Tris-HCl (pH 7.4). 10 μL of this diluted solution was mixed with 10 μL of the fusion protein solution containing endothelin A receptor prepared in (3) above, and incubated at 37 ° C. for 3 hours.

After the incubation, the reaction solution was subjected to gel filtration using the same TSKgel G4000SWXL as in (3) above, and the target fusion protein fraction was recovered. Thereby, the conjugate of the fusion protein and the compound could be separated from the compound library. TCA was added to the collected fraction to precipitate the protein, and the supernatant fraction (containing only the compound) was collected. The supernatant fraction was combined with reverse phase chromatography using a C18 column and mass spectrometry (LC / MS / MS) to obtain a mass spectrum. As a control experiment, the same operation was performed using only a chaperonin conjugate containing no target protein instead of the fusion protein, and a mass spectrum was obtained. By comparing these difference spectra, the recovered low molecular weight compound was identified as BQ123.
From the above, it was shown that a compound that specifically binds to the endothelin A receptor, which is a hydrophobic target protein, can be efficiently screened.

Table 1 shows the base sequences and restriction enzyme sites of each primer used in this example.

It is explanatory drawing which shows the structure of the principal part of pTrc (GV) 7TC2H.

Claims (7)

A method for screening a compound that specifically binds to a hydrophobic target protein, comprising the following steps (1) to (3):
(1) contacting the target protein and chaperonin fusion protein with a compound library containing a plurality of types of compounds, and binding a desired compound to the target protein portion of the fusion protein;
(2) separating the conjugate of the fusion protein and the compound from the compound library;
(3) identifying a compound bound to the fusion protein;
A method for screening a compound, comprising:   The method for screening a compound according to claim 1, wherein the target protein is a membrane protein.   In the step (2), the conjugate is separated by at least one technique selected from chromatography, molecular sieving, filter separation, and precipitation. Screening method.   The compound screening method according to any one of claims 1 to 3, wherein the compound is identified by mass spectrometry in the step (3).   The method for screening a compound according to any one of claims 1 to 4, wherein the fusion protein is a fusion protein of a target protein and a chaperonin subunit conjugate.   The method for screening a compound according to any one of claims 1 to 5, wherein the chaperonin forms a single layer structure.   A kit for use in the method for screening a compound according to any one of claims 1 to 6, comprising the fusion protein and the compound library.

Images