KR100437991B1 - Crystal structure of the complex of human phosphodiesterase 4d with inhibitor thereof and method for screening regulator of human phosphodiesterase 4d - Google Patents

Abstract

PURPOSE: 3D-Dimensional crystal structure of a complex of human phosphodiesterase 4D and its inhibitor, and a method for identifying a material regulating an activity of human phosphodiesterase 4D using the same are provided. The complex has an easy X-ray analysis and improved crystallinity, so that the crystal structure can be useful for treatment of asthma and immunological disease associated with human phosphodiesterase 4D. CONSTITUTION: The 3D-dimensional crystal structure of a complex of human phosphodiesterase 4D and adenine is provided, wherein the atom coordinates of the 3D-dimensional crystal structure of the complex are listed in table 2(b). The method for identifying a material regulating an activity of human phosphodiesterase 4D comprises the steps of: providing the 3D-dimensional crystal structure of a complex of human phosphodiesterase 4D and adenine by using X-ray crystallography; providing a candidate material regulating the activity of human phosphodiesterase 4D; and verifying the binding of the candidate activity regulating material to the adenine binding site of human phosphodiesterase 4D.

Description

CRYSTAL STRUCTURE OF THE COMPLEX OF HUMAN PHOSPHODIESTERASE 4D WITH INHIBITOR THEREOF AND METHOD FOR SCREENING REGULATOR OF HUMAN PHOSPHODIESTERASE 4D}

The present invention relates to crystallization of a complex in which a human phosphodiesterase 4D, which is a target protein related to asthma and immune diseases, and an inhibitor is combined, and a three-dimensional structure of the crystal, and to modulate the activity of human phosphodiesterase 4D using the same. A method of identifying / designing a substance.

Cyclic nucleotides are intracellular secondary messengers that play an important role in many physiological phenomena. Intracellular concentration levels of these nucleotides are regulated by receptor-associated enzymes (eg, adenylyl cyclase and guanylyl cyclase) at the synthesis stage and by a family of enzymes known as phosphodiesterases at the degradation stage. do. Phosphodiesterases hydrolyze cAMP and cGMP to produce AMP and GMP. Eleven phosphodiesterase genes are known and more than 50 isoforms have been found in various tissues. These isotypes are expressed and regulated differently according to cell types, and their presence sites, substrate selectivity, reaction rate, and reactivity with accelerators or inhibitors appear to be different. In addition, phosphodiesterases are clinical target proteins such as retinal breakdown, congestive heart failure, depression, asthma, erectile dysfunction, inflammation, etc. TJ, Am J Respir Crit Care Med , 157: 351-370 (1998); Souness JE, et al., Immunopharmacol , 47: 127-162 (2000); Barnette MS and Underwood DC, Curr Opin Pulm Med , 6: 164 -169 (2000); Nell H, et al., Am J Respir Crit Care Med , 161: A 200 (2000); Timmer W, et al., Am J Respir Crit Care Med , 161: A505 (2000); and Torphy TJ, et al., Pulm Pharmacol Ther , 12: 131-135 (1999)).

In 11 known phosphodiesterase populations, cAMP specific phosphodiesterase 4 has four isoforms of A to D, and each isoform differs in expression depending on the site of the body, and the N-terminal region excluding the active domain Although slightly different in length, each homozygous active site has the same structure and amino acid residues (Xu RX, et al., Science , 288: 1822-1825 (2000); and Lee ME, et al., FEBS Letters , 530: 53-58 (2002)).

Phosphodiesterase 4 is particularly expressed in inflammatory cells, immune cells, and bronchial smooth muscle, and inhibition of phosphodiesterase 4 in these cells blocks trafficking and differentiation, and inflammatory mediators, cytokines And production of reactive oxygen species is reduced. Inhibitors of phosphodiesterase 4 generally have the effect of anti-inflammatory and bronchial dilatation in animal models, and in clinical trials have been shown to be effective in treating asthma, chronic obstructive pulmonary disease (COPD) and atopic dermatitis. (Barnette MS and Underwood DC, Curr Opin Pulm Med , 6: 164-169 (2000); Torphy TJ, et al., Pulm Pharmacol Ther , 12: 131-135 (1999); Hanifin JM, et al. J Invest Dermatol , 107: 51-56 (1996); and Souness JE, et al., Immunopharmacol , 47; 127-162 (2000). However, several phosphodiesterase 4 inhibitors have side effects such as dizziness and vomiting, making it difficult to apply them clinically (Horowski R and Sastre-y-hernandez M, Current Therapeutic Res , 38:23 (1985); Robichaud A). , et al., Neuropharmacology , 38: 289-297 (1999); and Barnette MS, Prog Drug Res , 53: 193-229 (1999).

Phosphodiesterase 4 is a divalent cation dependent hydrolase. Protein three-dimensional structural studies of phosphodiesterase 4B have been characterized by Robert et al. Of GlaxoSmithklein (Robert X, et al., Science , 288: 1822-1825 (2000)). This structure provides information about the steric shape of the active domain, the action of the enzyme and the presence of metal cations in the active site. Two divalent cations are bound to the common amino acids of these phosphodiesterases, one (M1) coordinates His238, His274, Asp275 and Asp392, and the other (M2) water molecules to Asp275 and M1 Weak coupling through. These metal ions have a high affinity with cAMP, chelating the phosphate group of cAMP to increase the electron affinity of the phosphate group to improve the reactivity.

Phosphodiesterase inhibitors studied so far include Cylomilast and Roflumilast from GlaxoSmithKline, BAY 19-8004 from Bayer and YM- from Yamanouchi Pharmaceuticals. 976 and the like. The efficacy of these compounds has been verified in animal experiments and clinical trials, and the better the drug efficacy and selectivity, the less side effects such as dizziness and vomiting. Thus, there is a need for the development of new phosphodiesterase 4 inhibitors with improved efficacy than these (Torphy TJ, Am J Respir Crit Care Med , 157: 351-370 (1998); Souness JE, et al. , Immunopharmacol , 47: 127-162 (2000); Barnette MS and Underwood DC, Curr Opin Pulm Med , 6: 164-169 (2000); Nell H, et al., Am J Respir Crit Care Med , 161: A200. (2000); Timmer W, et al., Am J Respir Crit Care Med , 161: A505 (2000); and Torphy TJ, et al., Pulm Pharmacol Ther , 12: 131-135 (1999).

In order to quickly and effectively discover such drugs having good efficacy and selectivity, a method of identifying a three-dimensional structure of a protein using crystallization or the like and discovering regulatory substances based on the structure of the protein are useful methods. Currently, phosphodiesterase 4 has been shown to have a protein-only structure to which no inhibitor binds (Robert X, et al., Science , 288: 1822-1825 (2000), which alone indicates the specific binding mechanism of the inhibitor and cAMP). It is unknown that the conformation of amino acid residues interacting with the inhibitor also depends on conjecture.

Therefore, the present inventors have made a complex in which an inhibitor is bound to phosphodiesterase 4 and elucidates the crystal structure of the complex, thereby revealing a binding mechanism between phosphodiesterase 4 and an inhibitor, and amino acid residues involved in binding and these To determine the three-dimensional shape of the new inhibitors will be utilized. In addition, information on whether the structure of the active site is changed or maintained by the binding of the inhibitor can be obtained through complex structure studies.

In the present invention, zardaverine used as an inhibitor of phosphodiesterase 4D is 6- (4- (difluoromethoxy) -3-methoxyphenyl) -3 (2H) -pyridazinone (6- Compound having the structure of (4- (difluoromethoxy) -3-methoxyphenyl) -3 (2H) -pyridazinone), is an inhibitor of phosphodiesterases 3 and 4. The compound has a 4- (difluoromethoxy) -3-methoxyphenyl group of the catechol type, which is an important binding site for most inhibitors of phosphodiesterases. It provides important information for studying the binding mechanism of podiesterase 4 and inhibitors and finding new inhibitors.

In addition, adenine (adenine) used as an inhibitor of phosphodiesterase 4D in the present invention is part of the structure of cAMP, which is a substrate of phosphodiesterase 4, and important information on substrate binding of cAMP specific phosphodiesterase To provide.

The present inventors crystallize a complex of phosphodiesterase 4D and an inhibitor that binds to its active site, and identify the three-dimensional structure of the crystal by x-ray crystallization to modulate the activity of phosphodiesterase 4D. The present invention has been completed by establishing a method of identifying and devising a substance.

It is an object of the present invention to provide a method for crystallizing a complex in which a new antibiotic target protein, human phosphodiesterase 4D, and an inhibitor are bound.

It is another object of the present invention to provide a three-dimensional structure of a complex crystal in which phosphodiesterase 4D and an inhibitor are crystallized by the method through x-ray crystallography.

It is still another object of the present invention to provide a method for discovering an activity modulator of phosphodiesterase 4D using a three-dimensional structure of a complex crystal in which phosphodiesterase 4D and an inhibitor are bound.

Figure 1 is a schematic of the physiological mechanism of phosphodiesterase 4 (Phosphodiesterase 4),

Figure 2 shows the structure of the complex conjugated phosphodiesterase 4D and the inhibitor zadaverine (zadaverine),

Figure 3 shows the structure of the complex conjugated to the phosphodiesterase 4D and the inhibitor adenine (adenine),

Figure 4 shows the structure of the complex conjugated phosphodiesterase 4D and the inhibitor jadaberin as the surface structure of the protein and the skeleton of the inhibitor,

5 shows an electron density map of a complex in which phosphodiesterase 4D and an inhibitor jadaberin are bound,

6 and 7 are flow charts illustrating the inventive method of using a three-dimensional structure of a complex in which a phosphodiesterase 4D is combined with an inhibitor to identify regulatory candidates that modulate the activity of phosphodiesterase 4D. to be.

In accordance with the above object, the present invention is characterized in that the human phosphodiesterase 4D and the inhibitor-bound complexes are crystallized by a hanging-drop vapour diffusion method, characterized in that the human phosphodiesterase 4D It provides a method for crystallization of a complex in which an inhibitor is combined.

According to this another object, the present invention provides a three-dimensional structure of a complex in which human phosphodiesterase 4D and an inhibitor are bound through x-ray crystallization.

In accordance with another object, the present invention provides a method for identifying or designing a substance capable of modulating the activity of human phosphodiesterase 4D.

Hereinafter, the present invention will be described in detail.

For the crystallization of human phosphodiesterase 4D, any portion of the protein that exhibits high fluidity should be predicted and removed in advance. If there is a part showing liquidity, crystals are not well formed, so removing them is very important to increase the probability of success of crystallization. Thus, the human phosphodiesterase 4D used in the present invention contains amino acid 510 serine from amino acid 183 threonine (N-terminus) so that it contains all the active domains of the enzyme without including the part showing fluidity. Human phosphodiesterase 4D up to (C-terminus) is used and a GST (Glutathione S Transferase) protein is bound to the N-terminus to facilitate purification, and this GST protein portion is removed in the final step. .

Human phosphodiesterase 4D is expressed by known genetic engineering methods.E. Coli amplified a gene encoding the amino acid sequence by PCR, cloned it into a GST fusion expression vector, and transformed with the cloning vector. It can be obtained by being expressed by.

The phosphodiesterase 4D thus obtained must undergo a purification step by ion exchange resin column, gel filtration chromatography, etc. in a known manner.

After purification of phosphodiesterase 4D as described above, it is crystallized.

There are various methods of crystallizing proteins, but in the present invention, a hanging-drop vapour diffusion method (Jancarik, J. et al., J. Appl. Cryst ., 24 , 409-411 (1991) The complex in which the human phosphodiesterase 4D and the inhibitor are bound is crystallized as follows.

When small droplets of mother liquor and a much larger reservoir solution coexist in a confined space with a 1: 1 ratio of protein solution and reservoir solution, water or other volatiles move between them. .

Under supersaturated protein solution conditions, in these thermodynamic metastable states, the protein precipitates as the concentration of precipitant increases due to the migration of water or other volatiles from the initial state of the mother liquor. Becomes The precipitant serves to reduce the solubility of the concentrated protein solution. Proteins gather together to form crystals to reduce the relative adsorption layer around them.

The reservoir solution is a mixture of precipitants, buffers, salts, detergents, etc. In general, the protein solution and the reservoir solution are mixed in a ratio of 1: 1 to prepare drops of mother liquor.

The prepared drops are dropped on a glass slide surface coated with silicone and the slide is sealed on a plate containing a reservoir solution. Initially, the concentration of protein in the droplets differs from that of the reservoir solution so that the protein does not exist in the crystalline state. When left in this sealed state, equilibrium is gradually achieved, in which crystals are formed under specific conditions according to the principles described above.

In this drop-mixed vapor equilibrium method, the pH, temperature, and type and concentration of precipitants, salts, buffers, and detergents of the reservoir solution are selected according to the type of protein, and in some cases, they are very important factors in the crystal formation of proteins. Becomes

The protein solution of the present invention is prepared by adding, to a solution of phosphodiesterase 4D, jadaberin or adenine of the following structure, an inhibitor that binds to the active site of human phosphodiesterase 4D:

The reservoir solution may comprise a precipitant and a buffer. It is preferable to use cacodylate buffer as the buffer, and the concentration of the buffer is 0.05 M to 0.20 M, preferably 0.10 M. In addition, the pH of the reservoir solution is 6.0 to 7.0, preferably pH 6.5.

As a precipitant for precipitating proteins, ammonium sulfate or polyethylene may be used, and polyethylene 8000 is preferable. The concentration of precipitant is 5% to 25%, preferably 11%.

The reservoir solution may also include sodium chloride, potassium chloride, citrate, lithium ammonium, calcium chloride salts or magnesium acetate salts, preferably magnesium acetate salts, as salts used to promote crystal formation. The concentration of the salt is 0.5 M to 0.8 M, preferably 0.6 M.

Protein solution and reservoir solution are generally mixed in a ratio of 1: 1 to make droplets and then contacted to the glass slide surface.

Crystal formation reaction temperature is 4-30 degreeC, Preferably it is 25 degreeC, and reaction time is 1 day-1 month, Preferably 1 week-2 weeks.

In addition, due to the nature of protein crystals, crystals exposed to high energy of x-rays have a shorter lifespan and weaker data, which affects structural analysis. can do.

This is a method of cooling liquid nitrogen to a temperature of 100 K and continuously exposing it during the experiment, which is necessary to continuously acquire data of a three-dimensional structure.

In the present invention, it is possible to effectively protect the human phosphodiesterase 4D crystals by liquid nitrogen cooling with a solution for fast cooling containing Tris buffer, calcium chloride salt, polyethylene glycol 8000, and paratone N.

In the solution for high speed cooling, the concentration of the cacodylate buffer is 0.05 to 0.20 M, preferably 0.10 M, and the concentration of the magnesium acetate salt is 0.5 M to 0.8 M, preferably 0.6 M. Fast nitrogen cooling is carried out at a temperature of 100 K.

According to another object of the present invention, there is provided a crystal structure of human phosphodiesterase 4D-zadaberin and human phosphodiesterase 4D-adenine complex.

In order to determine the three-dimensional structure of the complex crystal in which the human phosphodiesterase 4D and the inhibitor are obtained, the diffraction pattern is obtained using an x-ray image plate, and phase information is obtained through structure substitution. Get

A three-dimensional structure is obtained by preparing an electron density map from the x-ray diffraction pattern and phase information of the complex in which the human phosphodiesterase 4D and the inhibitor are combined, and then deriving atomic coordinates therefrom.

The collection of data using x-rays can be divided into methods using home laboratories and radiation (Synchrotron), depending on the source of the x-rays.

The use of radiated light enables crystals with crystal sizes as small as 50 μm, and data can be collected at multiple wavelengths as well as one wavelength, resulting in a quick structure.

In the present invention, diffraction data was obtained using the emitted light, and the spatial group of crystals was P2 ₁ 2 ₁ 2 ₁ , rectangular system; a = 97.3, b = 164.6, c = 325.5 Hz, and a data resolution of 2.9 Hz was obtained.

The phase information can be obtained through Multiple Isomorphous Replacement, Multiwavelength Anomalous Dispersion, and Molecular Replacement.

First, the heavy metal substitution method is to replace various crystals with heavy metals, collect data, analyze the information, and obtain phase information.

Secondly, multi-wavelength analysis is widely used in the last two to three years to obtain data after collecting data by using specific metals or atoms in the crystal instead of heavy metals and using the atomizer of the atoms at various wavelengths. have. In other words, the amino acid Met is replaced with se-Met from a single crystal using molecular biological method without the hassle of collecting data from multiple crystals, so that data can be easily obtained using this se atom. However, this method can only be obtained from radiated light.

Third, the structural substitution method solves the phase problem from similar structures already known, which is widely used as the known structures increase. After each structure is obtained from the data, a refinement process is performed to ensure that this data and our model fit as closely as possible. This process is carried out using a variety of programs (CCP4, O, Quanta, CNS, etc.) and standardization of each angle and coupling length is required. The process of fitting the density map is repeated. In the analysis phase after structural refinement, various information is inferred and interpreted from the structure. In this analysis phase, the mechanism of action based on the structure can be studied. It is the basis for obtaining branch information. In addition, the structure of the complex with the inhibitor for the protein can directly identify the relevant residues, providing important information for the next step in the study of the inhibitor.

In the present invention, the phase information was obtained by using a structural substitution method, and the calculation of the structural substitution method was performed using the AmoRe program (Navaza, J., Acta Crystallogr. , A50 , 157-163 (1994)), and refinement of the structure. Was performed with the CNS program (Brunger, AT. Et al., Acta Cryst., D54 , 905-921 (1998)).

The resulting phase information is used to calculate the electron density function, which has a distribution of 0 to 360 ° at an angle given one at each of 740,000 diffraction points.

The structure of the complex of the human phosphodiesterase 4D and the inhibitor that minimizes energy is derived through the Cache (Fujitsu) program to select the optimal structure that best matches the electron density.

The three-dimensional structural model of the complex in which the human phosphodiesterase 4D and the inhibitor are bound after the refinement operation comprises 12 human phosphodiesterase 4D active domains, each containing one zinc, magnesium, dimethylarsenate ( dimethylarsenate), and one molecule of jadaberin or adenine.

An atomic model of the complex in which the human phosphodiesterase 4D and the inhibitor are combined as shown in Table 2 from the three-dimensional structure of the human phosphodiesterase 4D-zadaberine and the human phosphodiesterase 4D-adenine complex Get

In general, the active site of human phosphodiesterase 4D can be predicted, but in the present invention, through the structure of the complex in which the human phosphodiesterase 4D and the inhibitor are bound, the three-dimensional structure of the active site that actually binds to the inhibitor is identified. It was.

As a result, the active site of phosphodiesterase 4D to which the inhibitor binds is F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, N306, D369, E436, Q440, Amino acid residues of S305, C455 and S452, more specifically F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, N306, D369, E436, Q440, It was found to contain amino acid residues of S305, C455, S452, N418, P419, Y426, D264, T430, L791 and L943.

The present invention provides a method for identifying an activity modulator that inhibits the function of phosphodiesterase 4D using the three-dimensional structure of the complex in which the phosphodiesterase 4D and the inhibitor are obtained.

Methods for identifying modulators that bind to human phosphodiesterase 4D include: 1) x-ray crystallogrphy using x-ray crystallogrphy 3 of the conjugated conjugate of human phosphodiesterase 4D Providing a dimensional structure; 2) providing a candidate for modulating activity of human phosphodiesterase 4D; And 3) confirming that the activity control candidate substance binds to the active site of human phosphodiesterase 4D.

Step 1) provides a three-dimensional structure of the complex in which the human phosphodiesterase 4D and the inhibitor are identified as described above. This step provides a three-dimensional structure that includes all or part of the atomic coordinates of Table 2, which is F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, Amino acid residues of N306, D369, E436, Q440, S305, C455 and S452, more specifically F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, Three-dimensional structure with an active site comprising amino acid residues of N306, D369, E436, Q440, S305, C455, S452, N418, P419, Y426, D264, T430, L791 and L943, or represented by the atomic coordinates of Table 2 It includes.

In step 2), a candidate substance for controlling activity of human phosphodiesterase 4D is provided. The candidate substance of the present invention analyzes the active site structure of the phosphodiesterase and includes a group of proteins having similar active sites. After enclosing, it includes a library of compounds capable of selectively binding thereto (Crystalgenomics, Korea).

In step 3), a virtual screening ( in silico HTS) method is performed to confirm whether the control candidate substance provided in step 2) binds to the active site of phosphodiesterase 4D (Sung-gu, Fine Chemical 2001 , 59:49 (2001). That is, the compound library provided in step 2) is subjected to a fast binding test on the three-dimensional structure of phosphodiesterase 4D on a computer using the Ludi program of Acelys. As a result of the fast binding test, ligands can be prioritized using energy scoring or self-developed scoring methods in Rudy.

The present invention also provides a method of designing a modulator that binds to human phosphodiesterase 4D through modification of the modulator identified by the above method in order to enhance the pharmacological effect of the modulator.

Methods of devising a modulator that binds to human phosphodiesterase 4D include: 1) providing a three-dimensional structure of a complex in which human phosphodiesterase 4D and an inhibitor are bound using x-ray crystallization; ; 2) providing a candidate for modulating activity of human phosphodiesterase 4D; And 3) confirming that the activity control candidate substance binds to the active site of human phosphodiesterase 4D; 4) modifying an activity modulating candidate that binds to the active site of human phosphodiesterase 4D to provide a modified candidate; And 5) confirming that the modified candidate substance binds to the active site of human phosphodiesterase 4D.

Step 1) provides a three-dimensional structure of the complex in which the human phosphodiesterase 4D and the inhibitor are identified as described above. This step provides a three-dimensional structure that includes all or part of the atomic coordinates of Table 2, which is F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, Amino acid residues of N306, D369, E436, Q440, S305, C455 and S452, more specifically F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, Three-dimensional structure with an active site comprising amino acid residues of N306, D369, E436, Q440, S305, C455, S452, N418, P419, Y426, D264, T430, L791 and L943, or represented by the atomic coordinates of Table 2 It includes.

In step 2), a candidate substance for controlling activity of human phosphodiesterase 4D is provided. The candidate substance of the present invention analyzes the active site structure of the phosphodiesterase and includes a group of proteins having similar active sites. After enclosing, it includes a library of compounds capable of selectively binding thereto (Crystalgenomics, Korea).

In step 3), a virtual screening method is performed to confirm whether the candidate substance provided in step 2) binds to the active site of phosphodiesterase 4D (Sung-gu, Fine Chemical 2001 , 59:49 (2001)). That is, the compound library provided in step 2) is subjected to a fast binding test on the three-dimensional structure of the phosphodiesterase 4D on a computer using the Rudy program of Acelis. As a result of the fast binding test, ligands can be prioritized using energy scoring or self-developed scoring methods in Rudy.

In step 4), the parent nucleus of the regulatory candidate substance identified as binding to human phosphodiesterase 4D to modify the activity regulation candidate substance is immobilized and the weak functional group is substituted with various weak functional group libraries. In general, the mother nucleus acts as a support for binding to the protein, and since the weak functional group directly interacts with the side chain of the protein, the drug may be improved through modification of the weak functional group. In order to modify the activity modulators, Rudy's Rudy program and the like can be used.

In step 5), virtual screening is performed in the same manner as in step 3) to confirm whether the modified candidate substance provided in step 4) binds to the active site of phosphodiesterase 4D.

Another method of designing a modulator that binds to human phosphodiesterase 4D is to provide a three-dimensional structure of a complex in which human phosphodiesterase 4D is combined with an inhibitor using x-ray crystallization. ; 2) generating a three-dimensional structure of a modulating material having a three-dimensional structure; And 3) confirming that the modulator produced in step 2) binds to the active site of phosphodiesterase 4D.

Step 1) provides a three-dimensional structure of the complex in which the human phosphodiesterase 4D and the inhibitor are identified as described above. This step provides a three-dimensional structure that includes all or part of the atomic coordinates of Table 2, which is F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, Amino acid residues of N306, D369, E436, Q440, S305, C455 and S452, more specifically F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, Three-dimensional structure with an active site comprising amino acid residues of N306, D369, E436, Q440, S305, C455, S452, N418, P419, Y426, D264, T430, L791 and L943, or represented by the atomic coordinates of Table 2 It includes.

Step 2) provides a virtual compound library by introducing a new parent nucleus and various drug functional groups based on a rational drug discovery method to generate a three-dimensional structure of a modulator. Asselis' Rudy program and the like may be used to produce an activity modulator.

In step 3), a virtual screening method is performed to confirm that the modulator provided in step 2) binds to the active site of phosphodiesterase 4D (Sung Goo, Fine Chemical 2001 , 59:49 (2001)). In other words, the compound library provided in step 2) was subjected to a fast binding test on the three-dimensional structure of phosphodiesterase 4D on a computer using the Rudy program of Acelis. As a result of the fast binding test, ligands can be prioritized using energy scoring or self-developed scoring methods in Rudy.

The present invention also provides a floppy diskette, a hard disk, and the like, which store data encoded by computer readable data of a three-dimensional structure of a complex in which a human phosphodiesterase 4D and an inhibitor combined above are identified. Such media further comprise amino acid residues of F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, N306, D369, E436, Q440, S305, C455 and S452. Specifically, F469, L416, M370, M434, I433, F437, M454, Q466, Y256, W429, M370, S371, E327, N306, D369, E436, Q440, S305, C455, S452, N418, P419, Y426, D264 And a medium having an active site comprising the amino acid residues of T430, L791, and L943, or storing a complex three-dimensional structure in which the human phosphodiesterase 4D and the inhibitor are bound by the atomic coordinates of Table 2. .

The present invention also provides a computer for providing a three-dimensional structure of a complex in which a human phosphodiesterase 4D and an inhibitor are bound, which computer comprises 1) the atomic coordinates or the x-ray diffraction pattern of Table 2. A device readable data storage medium including a data storage medium for coding data readable by the device; 2) available storage for processing data; 3) a central processing unit (CPU) connected with available memory and data storage media; And 4) a display device connected to the central processing unit.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely examples for the present invention, and the scope of the present invention is not limited thereto.

Reference Example 1. Expression and Purification of Human Phosphodiesterase 4D Gene

Based on the sequence information for human phosphodiesterase 4D, the human phosphodiesterase 4D (GenBank ID U02882) gene from the N-terminal amino acid 183 (Threonine) to the C-terminal amino acid 510 (serine) was selected. Synthesis was performed by polymerase chain reaction (PCR). The gene obtained from the PCR was cloned into the GST fusion expression vector pGEX4T3 (APbiotech) and transformed into the expression host E. coli BL21 (DE3) (Novagen Inc., USA).

The transformed E. coli strains were shaken for 12 hours in LB medium containing 100 μg / ml ampicillin, and then the 1 ml culture was transferred to 100 ml LB medium (including 100 μg / ml ampicillin) at room temperature. When the absorbance was about 0.5 at 600 nm, IPTG was added to a final concentration of 0.4 mM. Cell precipitates were harvested by centrifugation at 10,000 g for 2 minutes before and 12 hours after addition of IPTG. The cell precipitate was suspended in a solution of 50 mM Tris (pH 8.0), 0.4 M NaCl, 5 mM DTT to treat 0.2 mg / ml of lysozyme, and the cells were sonicated on ice on ice.

The cell mill was centrifuged to allow the supernatant to pass through a Q-sepharose (APbiotech) ion exchange resin, and the passthrough was adsorbed onto a glutathione-sepharose column (APbiotech). The adsorbed protein was eluted with a solution containing 5 mM glutathione, 50 mM Tris (pH 8.0), 0.4 M NaCl and 5 mM DTT, followed by treatment with 0.5% (w / w) thrombin overnight at 4 ° C. GST protein fused to diesterase 4D was removed. Phosphodiesterase 4D from which GST protein was removed was purified using Source Q anion exchange resin and Superdex-200 gel filtration chromatography (APbiotech).

Example 1. Crystallization of Human Phosphodiesterase 4D Complex

The human phosphodiesterase 4D protein obtained in the reference example was crystallized by the following drop mixed vapor equilibrium method.

A protein solution was prepared having a concentration of 70 mg / ml of human phosphodiesterase 4D in a solution containing 50 mM Tris, pH 8.0, 0.4 M NaCl and 5 mM DTT. Final protein concentration was determined by the Bradford method (Current Protocols in Protein Science, 3.4.10). In a protein solution concentrated at 70 mg / ml, jadaberin (Biomol) or adenine (sigma aldrich) was mixed at a concentration of 3 mM, respectively.

Crystallization conditions were searched step by step with final protein solution containing jadaberin or adenine and initial screen solution (Hampton research Inc.). After mixing 2 µl of protein solution and 2 µl of reservoir solution, crystal formation was performed after 2 to 20 days at 4 ° C and 22 ° C. The reservoir solution was a crystal screen I solution kit (hampton research) with 50 different compositions. Crystals with the best x-ray diffraction capability were obtained when a reservoir solution consisting of 0.1 M cacodylate buffer (pH 6.5) and 11% polyethylene glycol 8000 precipitant was used.

A drop of a mixture of 2 μl human phosphodiesterase 4D protein solution and 2 μL reservoir solution is dropped onto a glass slide coated with silicone, and the slide is placed on a plate containing 0.5 mL of the reservoir solution at a constant temperature of 22 ° C. Put it. Seed crystals formed after 5 days and grown to 0.05 × 0.05 × 0.1 mm in size one week later.

Example 2 Rapid Cooling Experiment of Human Phosphodiesterase 4D Complex Crystals

In order to solve the problem that the human phosphodiesterase 4D complex crystals obtained from Example 1 were derived upon direct x-ray exposure, human phosphodiesterase 4D crystals were cooled by high-speed nitrogen cooling as follows.

Various cooling solutions such as glycerol, sodium formate, ethylene glycol, sucrose, and paratone N were searched at various concentrations, and then optimum conditions of the fast nitrogen cooling method were obtained. 1 x 100 x 20 x 20 micron size crystals in 1 μl of a solution for fast cooling containing 30% ethylene glycol, 0.10 M cacodylate buffer (pH 6.5), 11% polyethylene glycol 8000, 0.6 M magnesium acetate and 5 mM DTT. Soaking in 100% Paraton N (hampton research) within seconds and taking it out showed that it best tolerates 100 K liquid nitrogen stream without harming human phosphodiesterase 4D complex crystals.

Crystals soaked in the cooling solution were harvested using a 0.3 mm nylon crystal recovery tool (hampton research) and immediately placed in a -150 ° C. nitrogen stream.

In addition, the water ring that occurs frequently during the experiment (a phenomenon in which water freezes when high-speed nitrogen cooling is incomplete) was not seen.

Example 3 X-Ray Data Collection and Analysis of Human Phosphodiesterase 4D Crystals Via Radiation Accelerator

Using the human phosphodiesterase 4D complex crystal obtained in Example 2 was performed to collect data in the A1 beam line of the Cornell High Energy Synchrotron Source. The data limit of the crystal was 2.8 mm 3, and the data was treated with DENZO and SCALEPACK (Otwinowski, Z. and Minor, W. Methods Enzymol., 276 , 461-472 (1997)). The results of crystal data collection and refinement are shown in Table 1.

Example 4 X-ray Data Interpretation and Structure Calculation of Human Phosphodiesterase 4D Complex Crystals

The structure of human phosphodiesterase 4D-zadaberin or human phosphodiesterase 4D-adenine complex is based on the structure of human phosphodiesterase 4B enzyme active domain (Xu, RX et al., Science, 288: 1822 -1825 (2000)) through the structural substitution method. Structural substitution method calculations were performed using the AmoRe program (Navaza, J., Acta Crystallogr. , A50: 157-163 (1994)). Phosphodiesterase 4B enzyme active domain structure was used as a search tool to determine the protein position and orientation in the new crystal environment. The diffraction data used had a resolution of 3.5 Hz.

Structural refinement was done using the CNS program (Brunger, AT. Et al., Acta Cryst., D54 , 905-921 (1998)). The first refinement was performed using the simulated annealing method of the CNS program. The simulation start temperature was 1500 ° C, and each step was performed by cooling 100 ° C to 25 ° C, and the R factor dropped from 48% to 27%. After 12 times of non-crystallographic symmetry averaging, a strong and continuous electron density was found near the metal bond site.

The final refinement was performed by using 20 cached low energy jadaberin structures using the Cache (Fujitsu) program and selecting the optimal structure that best fits the electron density (see FIG. 5). Final model obtained after the final refinement (relative three-dimensional position of all atoms except hydrogen in the protein) contains twelve human phosphodiesterase 4D enzymatic active domains, one zinc, magnesium, dimethylarsenate for each protein (Cacodylate) (dimethylarsenate (cacodylate)), and one molecule of jadaberine or adenine.

According to the method of the present invention, it is possible to obtain human phosphodiesterase 4D complex crystals which are easy to x-ray analysis and excellent in crystallinity, and the three-dimensional structure of the crystals is identified to determine the activity of human phosphodiesterase 4D. By identifying and designing a substance that modulates the protein, it can be effectively used for the development and research of therapeutic agents related to asthma and immune diseases related to human phosphodiesterase 4D.

Claims (18)

delete delete delete delete delete delete delete delete A computer readable medium storing an atomic model representing the three-dimensional structure of a complex of human phosphodiesterase 4D and adenine bound. delete delete The method of claim 9, A medium characterized in that the three-dimensional structure of the complex in which human phosphodiesterase 4D and adenine are bound is represented by the atomic coordinates of Table 2 (b). A method of identifying a modulator that binds to human phosphodiesterase 4D, comprising the following steps: 1) using x-ray crystallogrphy to provide a three-dimensional structure of the complex of human phosphodiesterase 4D and adenine; 2) providing a candidate for modulating activity of human phosphodiesterase 4D; And 3) confirming that the activity regulation candidate binds to the adenine binding site of human phosphodiesterase 4D. A method of designing a modulator that binds to human phosphodiesterase 4D, comprising the following steps: 1) using x-ray crystallization to provide a three-dimensional structure of the complex of human phosphodiesterase 4D and adenine; 2) generating a three-dimensional structure of a modulating material having a three-dimensional structure; And 3) confirming that the modulator produced in step 2) binds to the adenine binding site of phosphodiesterase 4D. A method of designing a modulator that binds to human phosphodiesterase 4D, comprising the following steps: 1) using x-ray crystallization to provide a three-dimensional structure of the complex of human phosphodiesterase 4D and adenine; 2) providing a candidate for modulating activity of human phosphodiesterase 4D; 3) confirming that the activity control candidate binds to the adenine binding site of human phosphodiesterase 4D; 4) modifying an activity regulation candidate that binds to the adenine binding site of human phosphodiesterase 4D to provide a modified candidate; And 5) Confirming that the modified candidate substance binds to the adenine binding site of human phosphodiesterase 4D. delete The method according to any one of claims 13 to 15, Wherein in step 1) the three-dimensional structure of the complex in which the human phosphodiesterase 4D and adenine are bound is represented by the atomic coordinates of Table 2 (b). A computer for providing a three-dimensional structure of a complex in which human phosphodiesterase 4D and adenine are bound, comprising the following device: 1) a device readable data storage medium comprising a data storage medium for coding data readable by a device comprising atomic coordinates or x-ray diffraction patterns of Table 2 (b); 2) available storage for processing data; 3) a central processing unit (CPU) connected with available memory and data storage media; And 4) Display connected to the central processing unit.