JP2005170832A - Crystal of interleukin 6 having new crystal structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable cocrystallization of interleukin 6 and its antagonistic inhibitor, and furthermore enable the search, evaluation, and designing of an antagonistic inhibitor of interleukin 6. <P>SOLUTION: A crystal of interleukin 6 having a new crystal structure and its crystallization method are provided. A method of screening a compound which forms a complex by the interaction with the interleukin 6 is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a crystal of interleukin 6 having a novel crystal structure and a crystallization method thereof. The present invention further relates to a method for screening a compound that interacts with the interleukin 6 to form a complex.

Cytokines are proteinaceous signaling molecules that act as various intercellular signaling in the growth and differentiation of animal cells. Cytokines are characterized by, for example, the fact that they exhibit physiological activity only by binding to specific receptors on the cell surface, the presence of cytokine networks, and originally contribute to the maintenance of homeostasis in vivo. However, it is also deeply related to the mechanism of action of the disease state. That is, when a cytokine is produced, the cell is activated by binding to a specific receptor corresponding to the cytokine, and a physiological activity peculiar to each cytokine is expressed. In addition, there is a linkage called a cytokine network in which when one cytokine is produced, another cytokine is produced next. Furthermore, in the living body, a positive regulatory mechanism and a negative regulatory mechanism coexist in cytokine production and operate in a balanced state under normal conditions, but when this balance is lost, abnormal cytokine production states appear, and various Induce the pathological condition. Therefore, there is a need for inexpensive treatments that increase the quality of life (QOL) for many diseases caused by increased cytokine function, which contributes not only to individual patients but also to the domestic economy through the reduction of medical finances and global insurance. Expected to make a significant contribution to policy.
Nobuyuki Miyasaka, “Approach from the Latest Medicine (Volume 1) Cytokines”, Medical View, May 1991, p.1-4.

Interleukin 6 (hereinafter abbreviated as IL-6) is a kind of cytokine that is a glycoprotein isolated as a B cell stimulating factor that acts on activated B cells to induce differentiation into antibody-producing cells. It is known that abnormal production of IL-6 is not limited to an abnormal immune response in a living body, but is involved in various diseases such as abnormal hematopoiesis, excessive inflammatory response, and tumor growth. Such diseases are roughly classified into neoplastic diseases and non-neoplastic diseases. Examples of the former include multiple myeloma (see, for example, Non-Patent Document 2), Crohn's disease, malignant lymphoma, renal cancer, prostate cancer, Kaposioma, and atrial myxoma. Examples of the latter include mesangial proliferative nephritis, vulgaris Psoriasis and rheumatoid arthritis (see, for example, Non-Patent Document 3).
Hirano, T. and Kishimoto, T. et al., Excessive production of interleukin 6 / B cell stimulatory factor-2 in rheumatoid arthritis. Eur. J. Immunol. Vol.18, p.1797-1801, 1988. Kawano, M. and T. Kishimoto et al., Autocrine generation and requirement of BSF-2 / IL-6 for human maltiple myelomas. Nature Vol.332, p.83-85, 1988.

  In vivo, IL-6 binds to interleukin 6 receptor (IL-6R), which is a receptor specific for IL-6, to form an IL-6-IL-6R complex. Subsequently, the complex is known to bind to a homodimer of gp130, a receptor common to the IL-6 family, to form a hexamer, in which case a signal is sent into the membrane.

On the other hand, it is no longer necessary to know the structure of biopolymers including proteins in detail in order to carry out today's life sciences. In particular, the structural information at the atomic level obtained by X-ray crystal structure analysis is the life of these molecules. It is indispensable for understanding functions in the body. The structural analysis of the relationship between IL-6 and IL-6R or gp130 has been published so far (see, for example, Non-Patent Document 4; however, in this report, the receptor structure is a part of the extracellular domain only. The structure is not complete, and each binding mode is being elucidated in detail, and further medical scientific research is expected in the future.
Martin J. Boulanger, Dar-chone Chow, * Elena E. Brevnova, K. Christopher Garcia et al., Hexameric Structure and Assembly of the Interleukin-6 / IL-6 _ Receptor / gp130 Complex, Science Vol.300, p.2101 -2104, 2003.

Protein crystallization methods (see, eg, Non-Patent Document 5) generally differ from those used in low molecular weight organic compounds and involve many physiological factors (eg, pH, temperature, salt concentration). It is required to keep it constant. In particular, since crystallization is realized in a state of a supersaturation point due to a decrease in solubility, a salt concentration due to an additive such as a precipitant becomes a particularly important factor.
Edited by Satoshi Nakajima et al., “New Basic Biochemical Experimental Method (Volume 5) Higher-order Structure and State Analysis”, Maruzen Co., Ltd., September 1989, p.180-217).

  The precipitant used in the protein crystallization method is roughly classified into an inorganic salt, an organic solvent, and polyethylene glycol (hereinafter abbreviated as PEG). First, inorganic salts include sulfates (eg, ammonium sulfate and sodium sulfate), citrates (eg, ammonium citrate and sodium salts), phosphates (eg, potassium phosphate and sodium salts), acetate salts (eg, Sodium acetate and ammonium salts) and nitrates (eg, ammonium nitrate salts). Examples of the organic solvent include 2-methyl-2,4-pentanediol (abbreviated as MPD), isopropanol, dioxane, acetone, and the like.

  PEG is a useful organic additive along with the inorganic salt ammonium sulfate. PEG is readily available in a variety of products with a wide range of average molecular weight fractions. For example, oily PEG 200 (molecular weight 200) to waxy PEG 20000 are known.

  Furthermore, the crystal structure analysis of the complex of the above-mentioned IL-6 with a protein including IL-6R or gp130, a peptide that is an antagonist, a small organic molecule, or the like, ie, a co-crystal, Provides basic structural information important for the treatment of diseases related to abnormal production of drugs and the development of pharmaceuticals. In particular, a so-called IL-6 antagonistic inhibitor that binds IL-6 reversibly and competitively to the IL-6 receptor and inhibits the binding of IL-6 to the IL-6 receptor; The structural information of the co-crystal with IL-6 makes it possible to develop a simple screening method for searching, evaluating and designing a novel IL-6 antagonist. The co-crystal is obtained by immersing the protein crystal in a solution of the peptide or the like and allowing the peptide or the like to enter a predetermined site in the protein crystal. In this case, the conditions of the cocrystallization solution must usually be the same as the conditions used for crystallization of these proteins. There are several reasons for this, but one is that the IL-6 crystal dissolves or breaks under the influence of the surrounding salt if it is performed under conditions different from the crystallization conditions. It is done.

  Here, generally, the interaction between a protein and a protein (including a peptide or the like) is a bond such as a van der Waals interaction, a hydrogen bond, and an electrical interaction (that is, a Coulomb interaction). Therefore, co-crystallization under high salt concentration conditions, for example, because the salt of the surrounding environment used as a precipitant can diffuse throughout the protein crystal and bind to the protein, or the interaction between proteins is inhibited, or The peptide was eluted and precipitated due to the high salt concentration, which was very limited. Therefore, in order to achieve co-crystallization between proteins, it is essential to achieve crystallization of the proteins themselves under a low salt concentration. Furthermore, from the viewpoint of drug discovery, it can be said that it is poor in practicality unless it is information obtained by cocrystallization under the same low salt concentration conditions as in vivo.

As described above, the mechanism of action of IL-6-related diseases has been reported from a biochemical standpoint, but there are many unclear parts as a dependency from the structural standpoint. So far, X-ray crystal structure analysis of IL-6 (see, for example, Non-Patent Document 6) has been reported. However, the crystals of IL-6 were obtained using an inorganic salt such as ammonium sulfate as a precipitant at a high salt concentration. Therefore, in the conventional crystallization method, it has been difficult to search for, evaluate, and design an IL-6 antagonist by subsequently co-crystallizing IL-6 and a peptide.
Somers, W. and Seehra, JS et al., 1.9A crystal structure of interleukin 6: implications for a nevel mode of receptor dimerization and signaling., EMBO J 16, pp. 989 1977.

  The search, evaluation and design of IL-6 inhibitors establish conditions for crystallization of IL-6 under low salt concentration conditions that allow co-crystallization of IL-6 and IL-6 antagonist inhibitors This is very important. However, at present, a method for crystallizing IL-6 under a low salt concentration condition is not known.

  The present inventors succeeded in crystallizing IL-6 under low salt concentration conditions. The present inventors also conducted X-ray structural analysis on the obtained crystal, elucidated its three-dimensional structure, and found that the crystal itself was a crystal of IL-6 having a novel crystal structure.

The present invention is a tetragonal crystal of interleukin 6, wherein the unit cell constants are a = 49.28 ± 0.2%, b = 112.70 ± 0.3%, c = 32.54 ± 0.2%. The gist is a crystal having a space group of P2 ₁ 2 ₁ 2.

  The gist of the present invention is also a crystallization method of interleukin 6 characterized in that a mixture of polyethylene glycol 8000 and 1000 is used as a precipitating agent in the crystallization of interleukin 6 by the vapor diffusion method.

The present invention is a screening method for a compound that interacts with interleukin 6 to form a complex,
a) dissolving a candidate compound in a solution of a mixture of polyethylene glycol 8000 and 1000 as a precipitant, and then immersing the interleukin 6 crystals of claim 1 in the solution; and
b) analyzing whether the crystal is a co-crystal of the interleukin 6 and the candidate compound;
The gist of this screening method is also included.

The present invention further provides a screening method for a compound that interacts with interleukin 6 to form a complex,
a) dissolving interleukin 6 and a candidate compound in a solution of a mixture of polyethylene glycol 8000 and 1000 as a precipitant;
b) obtaining a crystal of the interleukin 6 and the candidate compound by a vapor diffusion method; and
c) analyzing whether the obtained crystal is a co-crystal of the interleukin 6 and the candidate compound;
The gist of this screening method is also included.

IL-6 Production Method For example, the production method of IL-6 in the present invention will be described below, but the method is illustrative and is not limited thereto.
A plasmid vector in which cDNA of interleukin 6 is incorporated into E. coli HB101 is incorporated by transformation, and 20 liters are cultured in a 30 liter culture apparatus. The microbial cells are collected, crushed by a high-pressure crusher, and collected as inclusion bodies in a centrifuge at 20 minutes at 7600G. Dissolve in 500 ml wash (20 mM, Tris-HCl, 30 mM NaCl, pH 7.5) and collect again by centrifugation at 8000 G for 15 minutes. The pellet is solubilized by standing in 6M guanidine hydrochloride pH 5.5 for 2 hours at room temperature. The guanidine concentration is lowered from 6 M to 0.6 M over 22 hours at 22 ° C., and the solution state is gradually changed to a final solution (0.6 M guanidine hydrochloride, 10 mM Tris-HCl, pH 8.5). By this work, interleukin 6 is refolded and becomes a protein having a normal structure. The obtained protein solution is passed through a column for gel filtration (Sephadex G-25) and eluted with an eluent (10 mM sodium acetate, pH 5.0) to recover interleukin 6. An appropriate amount of sodium acetate is added to the eluate to reach 250 mM pH 5.0, which is passed through a cation exchange column (CM-Sepharose FF) and eluted with a sodium acetate concentration gradient. The eluate is adjusted to pH 4.0 with formic acid, passed through a reverse phase HPLC column and eluted with an elution buffer (0.1 M sodium formate, pH 6.0) with a gradient of acetonitrile. Thereafter, it is further subjected to gel filtration (Sephadex G-25M) twice. The first elution buffer uses 20 mM acetic acid 10% acetonitrile, and the second elution buffer uses 5 mM sodium acetate, pH 4.5. Then, it is passed through a cation exchange column CM-SepharoseFF. Equilibration is performed with 10 mM sodium acetate, pH 5.0 and eluted with a sodium chloride gradient. This was repeated three times, and finally passed through gel filtration HPLC (Superdex-75HR60 / 600) and eluted with 10 mM sodium citrate, pH 6.0, and 10 mM sodium phosphate, 8.7 mM sodium citrate, pH 7.0. Buffer substitution and concentration with buffer solution (Ejima, D., Watanabe, M. Sato, Y., Date, M., Yamada, N., Takahara, Y: High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli., Biotechnol Bioeng .; Vol. 62 (3): p. 301-10, 1999).

(2) IL-6 Crystal Production Method As a protein crystallization method that can be carried out in the present invention, a method well known in the art as a protein crystallization method can be used. The crystallization method includes, but is not limited to, (static) batch method, dialysis method, free interface diffusion method, vapor diffusion method and the like. In particular, in the crystallization method of the present invention, since the concentration of the precipitant can be finely adjusted by the concentration of the external liquid, vapor diffusion that can achieve a low salt concentration under the conditions of cocrystallization that continues as described above. The method is preferred. A hanging drop method and a sitting drop method are particularly preferable.

  The principle of the vapor diffusion method is as follows. First, a closed system space is created using a crystallization vessel. The in-container closed system is composed of an inner liquid (including droplets) containing protein and an outer liquid (including reservoir liquid). At that time, the salt concentration of the inner liquid and the concentration of the precipitating agent are previously set to concentrations lower than the supersaturation point, while the salt concentration of the outer solution and the concentration of the precipitating agent are set higher than the supersaturation point. Since there is a large osmotic pressure difference between the internal and external liquids, water vapor gradually moves from the internal liquid toward the external liquid, and the concentration of each component in the internal liquid is different from that of the external liquid through this vapor equilibrium. Ascend slowly until equilibrium is reached. As a result, it becomes possible to create a supersaturated state of the internal liquid, and the target crystal can be obtained.

For example, the crystallization method using the vapor diffusion method in the present invention will be described below, but the method is illustrative and is not limited thereto.
The crystallization method of the present invention using the vapor diffusion method is:
a) providing a solution of IL-6;
b) providing a precipitant solution containing a mixture of PEG 8000 and 1000 as precipitant;
c) mixing the IL-6 solution and the precipitant solution to obtain mixed droplets; and d) separately mixing the resulting mixed droplets and the reservoir solution precipitant solution obtained in b); The step of allowing the mixed droplet to stand until an IL-6 crystal grows in the droplet due to vapor equilibrium is included.

  In the hanging drop method, in the step of d), the mixed droplet obtained in the mixing step of c) is suspended on a solution reservoir holding a precipitant solution in a sealed container, and the solution The concentration of the precipitant in the reservoir is higher than the concentration in the mixed droplet.

  Further, in the sitting drop method, in the step d), the mixed droplet obtained in the mixing step c) is placed in a droplet stage above the solution reservoir that holds the precipitant solution in a sealed container. The concentration of the precipitant solution in the solution reservoir is higher than that in the mixed droplet.

  The precipitating agent used in the crystallization method of the present invention includes polyethylene glycol (abbreviated as PEG) having various average molecular weight fractions, and includes, for example, PEG 200 to 20000, but is not limited thereto. A mixture of PEG 8000 and 1000 is particularly preferred, and a weight ratio of about 1: 2 to about 2: 1 is acceptable, and a mixture having a weight ratio of 1: 1 is particularly preferred.

  Furthermore, the crystallization method of the present invention may further contain one or more buffering agents. Examples of the buffer include, but are not limited to, Tris (tris (hydroxymethyl) aminomethane) (for example, Tris sodium citrate) and sodium phosphate. Furthermore, the pH of the crystallization solution can be adjusted with the buffer, and other pH values are also contemplated by the present invention, but are preferably between 6 and 7.5.

The crystallization conditions by the hanging drop vapor diffusion method of the present invention will be exemplified.
Droplet composition:
2-10 μL of IL-6 10-20 mg / mL
Mixture of 1: 1 weight ratio of PEG 8000 and 1000 4-5%
Tris sodium citrate 0.5-20 mM (pH 7.0)
Sodium phosphate aqueous solution 0.1-20 mM
Reservoir composition:
Capacity 0.3-0.5mL
PEG 8000: 1000 weight ratio 1: 1 mixture 8-10%

  The concentration of the IL-6 solution in the droplets is acceptable in the range of about 1-20 mg / mL, and preferably in the range of 10-20 mg / mL. The concentration of the buffer is acceptable in the range of about 0.1 to 200 mM, preferably in the range of about 5 to 100 mM. By adding the buffer, the pH of the droplet is about 6 to 7.5. The range is acceptable. The drop volume is allowed to be about 1-10 μL. On the other hand, the volume of the reservoir liquid is allowed to be about 0.1 to 0.8 mL.

Crystallization is performed at 4 to 20 ° C., and good crystals are obtained after several days. The obtained crystal was a hexagonal plate and was found to be different from the crystal of the space group p3 ₁ 21 reported so far. For comparison, the crystallization conditions that have been reported so far (see reference 6 above) are that the concentration of IL-6 in the droplets is 15 mg / mL, and the reservoir solution contains a precipitating agent. PEG is not used, but high concentrations of salt (ie 1.8M ammonium sulfate, 300 mM potassium potassium tartrate, 100 mM sodium citrate) are used. Here, the salt in the droplets under the crystallization conditions of the present invention is a salt contained in a buffer that is usually used in the protein crystallization method, and includes salts such as sodium citrate and sodium phosphate. However, it is not limited to these. These salt concentrations are much significantly lower than the salt concentrations normally used as precipitants in protein crystallization methods. That is, the salt concentration normally used as a precipitating agent is about 100 mM to 2 M, whereas the acceptable salt concentration in the present invention is about 0.1 mM to 200 mM, and the salt is a low salt whose order is about 10 ² times. Crystallization was possible at the concentration. Thus, we succeeded in achieving crystallization of IL-6 at a significant low salt concentration.

(3) Method of X-ray structure analysis of IL-6 crystal For example, the method of the X-ray structure analysis of IL-6 crystal in the present invention will be described below, but the method is illustrative and limited to these. Is not to be done.
X-ray structural analysis was performed on the crystals of IL-6 obtained by the above crystallization method. The X-ray diffraction data of the single crystal can be collected using an X-ray generator equipped with a rotating counter-cathode or X-rays using emitted light. In the present invention, SPring8 Protein Research Beamline BL44XU is used as an apparatus. Next, the processing of diffraction spots was performed using Denzo (Max Science) as a processing program for X-ray diffraction data, and a resolution of 2.26 mm was obtained. The heavy phase multiple isomorphous substitution method, multiwavelength anomalous dispersion method, and molecular substitution method can be used to determine the initial phase (Jan Drenth [Principles of Protein X-ray crystallography], Springer-verlag Inc. (1994 )). Since the structural analysis data of IL-6 is known (see the above cited reference 6), the initial phase in the present invention was determined by using data (PDB No. 1ALU) by Somers et al. Registered in the PDB. A molecular replacement method was used. Next, after the initial phase was determined, an initial molecular model was constructed. In the construction, a model was created by a workstation using a graphics program. After the model was constructed, the structure was refined using CNS, a 3D structure refinement program that imposed stereochemical constraints. The resulting molecular model was modified using program O (Jones et al., Acta Crystallogr. A 47; 1991, 110-119).

  The present invention also relates to a method for screening a compound that interacts with IL-6 to form a complex. The screening method can be performed using the following two methods. That is, in the first method, a) a step of dissolving a candidate compound in a solution of a mixture of polyethylene glycol 8000 and 1000 as a precipitant and then immersing the IL-6 crystals obtained in the present invention in the solution; And b) analyzing whether the crystal is a co-crystal of the IL-6 and the candidate compound. Here, the step of obtaining the co-crystal of a) is performed according to the description in the above-mentioned method for producing IL-6 crystal. That is, the candidate compound is dissolved in a solution containing a buffer (eg, Tris) in the presence of a mixture having a weight ratio of PEG 8000 and 1000 as the precipitating agent of, for example, 1: 1, and then obtained in the above. The crystals of IL-6 are immersed. Here, the concentration of IL-6 and the candidate compound in the solution is allowed to be 0.05-1 mmol / mL for IL-6 and 0.5-10 mmol / mL for the candidate compound, respectively. An analysis is then performed to determine whether the crystals are the desired co-crystals of IL-6 and the candidate compound. Analytical methods include X-ray crystallographic methods, NMR methods, photomicrographs and the like. For example, the X-ray structure analysis method for the crystal can be performed according to a general X-ray structure analysis method. That is, X-ray diffraction data of the single crystal is collected, and then the initial phase is determined by a molecular replacement method using data obtained in the crystal structure analysis of the IL-6 crystal. After the initial phase is determined, an initial molecular model is constructed, and then the structure is refined using a refinement program. Finally, the crystal is analyzed to determine whether it is a desired co-crystal. By carrying out these analytical methods, it becomes possible to know the presence and the binding mode of the candidate compound.

  In the second screening method, a) a step of dissolving interleukin 6 and a candidate compound in a solution of a mixture of polyethylene glycol 8000 and 1000 as a precipitant; b) the interleukin 6 and the candidate compound by vapor diffusion C) and c) analyzing whether the obtained crystal is a co-crystal of the interleukin 6 and the candidate compound. That is, this method is characterized by directly obtaining a co-crystal of the interleukin 6 and the candidate compound.

  The steps a) and b) are carried out according to the description for the method for producing IL-6 crystals. That is, in the step a), the solution contains polyethylene glycol (abbreviated as PEG) having various average molecular weight fractions as a precipitant, and includes, for example, PEG 200 to 20000, but is not limited thereto. A mixture of PEG 8000 and 1000 is particularly preferred, and a weight ratio of about 1: 2 to about 2: 1 is acceptable, and a mixture having a weight ratio of 1: 1 is particularly preferred. The solution may further contain one or more buffering agents. Examples of the buffer include, but are not limited to, Tris (tris (hydroxymethyl) aminomethane) (for example, Tris sodium citrate) and sodium phosphate. Furthermore, the pH of the crystallization solution can be adjusted with the buffer, and other pH values are also contemplated by the present invention, but are preferably between 6 and 7.5. Further, as the vapor diffusion method of b), a hanging drop method and a sitting drop method are preferable. The concentrations of IL-6 and candidate compound in the droplet are allowed to be 0.05-1 mmol / mL for IL-6 and 0.5-10 mmol / mL for the candidate compound, respectively. The concentration of the buffer is acceptable in the range of about 0.1 to 200 mM, preferably in the range of about 5 to 100 mM. By adding the buffer, the pH of the droplet is about 6 to 7.5. The range is acceptable. The drop volume is allowed to be about 1-10 μL. On the other hand, the volume of the reservoir liquid is allowed to be about 0.1 to 0.8 mL. Next, an analysis is performed as to whether or not the obtained crystal is a desired co-crystal of IL-6 and the candidate compound. Analytical methods include X-ray crystallographic methods, NMR methods, photomicrographs and the like. For example, the X-ray structure analysis method for the crystal can be performed according to a general X-ray structure analysis method. That is, X-ray diffraction data of the obtained single crystal is collected, and then the initial phase is determined by a molecular replacement method using data obtained in the crystal structure analysis of the IL-6 crystal. After the initial phase is determined, an initial molecular model is constructed, and then the structure is refined using a refinement program. Finally, the crystal is analyzed to determine whether it is a desired co-crystal. By carrying out these analytical methods, it becomes possible to know the presence and the binding mode of the candidate compound.

(1) Production method of IL-6 crystal A crystallization method using a hanging drop method is described. IL-6 used was provided by Ajinomoto Co., Inc. First, an aqueous solution of IL-6 was prepared by dissolving IL-6 (21 mg) in an aqueous solution (pH 6.8) of Tris / sodium citrate 1 mM / sodium phosphate 1 mM. The final concentration of IL-6 was 21 mg / mL. Next, as a reservoir solution, PEG8000 (16 mg, manufactured by Hampton) and PEG1000 (16 mg, manufactured by Hampton) were added to water (1 mL) to prepare a 10% aqueous solution. This was used after being filtered through a 0.22 μ filter. A 24-well plate (manufactured by TPP) was used as a crystallization container. The cover glass was siliconized. In addition, silicone grease was applied to the mouth of the reservoir to bring the cover glass into close contact with each other to maintain vapor equilibrium. Next, on the cover glass, the above-mentioned IL-6 aqueous solution (2 μL) is added to the above-prepared reservoir solution (2 μL) and mixed with stirring, whereby a droplet having an PEG mixture concentration of 8% is obtained. Was prepared. After thorough mixing, the cover slip was gently inverted and suspended above the reservoir. And the crystal | crystallization was manufactured by standing this crystallization container in an incubator (20 degreeC). Crystals were formed after about 3 days, and crystals of IL-6 were obtained.

これまで報告されている結晶の空間群はＰ３_１２１の斜方晶系であるのに対し、本発明の結晶の空間群はＰ２_１２_１２の直方晶系を有していた。このことは、ＩＬ−６の新規な結晶系であることを示唆する。また、単位格子定数がａ＝４９．２８±０．２Å、ｂ＝１１２．７０±０．３Å、ｃ＝３２．５４±０．２Åであった。 (2) Knowledge obtained as a result of structural analysis of IL-6 crystal The structural analysis data of the crystal obtained in the present invention is summarized in Table 1 below. For comparison, structural analysis data described in the cited document 6 reported so far is also shown.
Table 1. X-ray crystal structure analysis data

The crystal space group reported so far is an orthorhombic system of P3 ₁ 21, whereas the crystal space group of the present invention has a tetragonal system of P2 ₁ 2 ₁ 2. This suggests that it is a novel crystal system of IL-6. The unit cell constants were a = 49.28 ± 0.2%, b = 112.70 ± 0.3%, and c = 32.54 ± 0.2%.

  The three-dimensional structure of IL-6 obtained as a result of the above structural analysis is shown in FIG. As shown in the figure, the main chain of the partial AB-loop from the 52nd to the 60th amino acid that has not been confirmed was successfully confirmed. Here, the AB-loop is a site called site III, which is known to be an important part in the interaction with gp130.

  According to the present invention, IL-6 was successfully crystallized under a low salt concentration. This enables co-crystallization of IL-6 and a compound that interacts with IL-6 to form a complex, and a screening method for a compound that interacts with IL-6 to form a complex. It has become possible. The compound that interacts with IL-6 to form a complex binds to IL-6 reversibly and competitively with the IL-6 receptor and binds to IL-6 and the IL-6 receptor. It is considered a so-called IL-6 antagonist that inhibits binding. Therefore, the IL-6 antagonist is useful for the treatment of diseases related to abnormal production of IL-6 and the development of pharmaceuticals. Therefore, it has become possible to develop a simple screening method for searching, evaluating and designing a novel IL-6 antagonistic inhibitor using the IL-6 crystals obtained in the present invention.

It is drawing which shows the crystal structure of the novel interleukin 6 crystal | crystallization obtained in this invention.

Claims (4)

A tetragonal crystal of interleukin 6 with unit cell constants of a = 49.28 ± 0.2%, b = 112.70 ± 0.3%, c = 32.54 ± 0.2%, and space A crystal, the group of which is P2 ₁ 2 ₁ 2.   A method for crystallization of interleukin 6, wherein a mixture of polyethylene glycol 8000 and 1000 is used as a precipitating agent in crystallization of interleukin 6 by a vapor diffusion method. A method for screening a compound that interacts with interleukin 6 to form a complex,
a) dissolving a candidate compound in a solution of a mixture of polyethylene glycol 8000 and 1000 as a precipitant, and then immersing the interleukin 6 crystals of claim 1 in the solution; and
b) analyzing whether the crystal is a co-crystal of the interleukin 6 and the candidate compound;
The screening method comprising: A method for screening a compound that interacts with interleukin 6 to form a complex,
a) dissolving interleukin 6 and a candidate compound in a solution of a mixture of polyethylene glycol 8000 and 1000 as a precipitant;
b) obtaining a crystal of the interleukin 6 and the candidate compound by a vapor diffusion method; and
c) analyzing whether the obtained crystal is a co-crystal of the interleukin 6 and the candidate compound;
The screening method comprising:

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