Three-dimensional structure of prostaglandin D synthase and utilization thereof

This application is a U.S. national stage of International Application No. PCT/JP02/09994 filed Sep. 27, 2002.

This application is a U.S. National Stage of International Application No. PCT/JP02/09994 filed Sep. 27, 2002.

The present invention relates to three-dimensional structures' of hematopoietic prostaglandin D synthase (which may refer to as “PGDS” hereinafter), and a method for designing PGDS inhibitor using the three-dimensional structures.

Prostaglandin D 2 (PGD 2 ) is synthesized in vivo by PGDS from prostaglandin H 2 (PGH 2 ). There are two types of PGDS, i.e., brain-type PGDS and hematopoietic PGDS. Hematopoietic PGDS absolutely requires glutathione for enzymatic reactions while brain-type PGDS also causes enzymatic reactions in the presence of thiol reagents other than glutathione.

PGD 2 is synthesized in the central nervous system by brain-type PGDS, and has functions of sleep induction, thermodepression, inhibition of secretion of corpus luteum hormone and control of responses to pain and odor. PGD 2 is synthesized in peripheral tissue by hematopoietic PGDS, and is known to have physiological functions including dilation of peripheral blood vessels, bronchoconstruction, and inhibition of platelet coagulation. It also acts as an allergic mediator released from mast cells (Lewis, R. A., Soter, N. A., Diamond, P. T., Austen, K. F., Oates, J. A. & Roberts, L. J. Prostaglandin D 2 generation after activation of rat and human mast cells with anti-IgE. J. Immunol., 129, 1627-1631 (1982)). It has been reported that allergic reaction is significantly reduced in knockout mouse lacking prostaglandin D 2 receptor protein (DP receptor) (Matsuoka, T., Hirata, M., Tanaka, H., Takahashi, Y., Murata, T., Kabashima, K., Sugimito, Y., Kobayashi, T., Ushikubi, F., Aze, Y., Yoshida, N., Honda, Y., Nagai, H. & Narumiya, S. Prostaglandin D 2 as a mediator of allergic asthma. Science, 287, 2013-2017 (2000)). There is another prostaglandin D 2 receptor (CRTH receptor) in Th2 lymphocytes, eosinophils, and basophils involved in allergic reactions, which promotes chemotaxis of those inflammatory cells (Hirai, H., Tanaka, K., Yoshie, O., Ogawa, K., Kenmotsu, K., Takamori, Y., Ichimasa, M., Sugamura, K., Nakamura, M., Takano, S., and Nagata, K., Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils and basophils via seven-transmembrane receptor CRTH2, J. Exp. Med., 193, 255-261 (2001)). The synthesis of prostaglandin D 2 in mast cells (Urade, Y., Ujihara, M., Horiguchi, Y., Igarashi, M., Nagata, A., Ikai, K. and Hayaishi, O. Mast cells contain spleen-type prostaglandin D synthase, J. Biol. Chem., 265, 371-375 (1990)) and human Th2 lymphocytes (Tanaka, K., Ogawa, K., Sugamura, K., Nakamura, M., Takano, S, and Nagata, K., Differential production of prostaglandin D2 by human helper T cell subsets, J. Immunol., 164, 2277-2280 (2000)) is made by hematopoietic PGDS.

If a compound capable of inhibiting the enzymatic activity of PGDS is discovered, it would become possible to control PGD 2 synthesis to obtain anti-allergic agents, sleep control agents and anti-obesity agents.

Recently, attempts to design medicines from the three dimensional structure of proteins have been made. However, no one has succeeded in determining the three dimensional structure of human hematopoietic PGDS. It is hence difficult to design an inhibitor of human hematopoietic PGDS from the three dimensional structure thereof. It is an object of the present invention to clarify the three dimensional structure of human hematopoietic PGDS to provide a method for designing an inhibitor of human hematopoietic PGDS using the three dimensional structure.

The inventors has succeeded in preparing crystals of complexes of human hematopoietic PGDS (which has as metal ion calcium ion (referred to as “calcium type” hereinafter) or magnesium ion (referred to as “magnesium type” hereinafter)), glutathione (GSH) which is a cofactor of PGDS, and analog of prostaglandin H 2 (PGH 2 ) which is a substrate of PGDS or several types of inhibitor of PGDS. The inventors have succeeded in clarifying the three dimensional structures of these complexes with crystallography techniques using X-ray diffraction methods for the first time.

The present invention is related to a complex of human calcium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1 and glutathione which has a three dimensional structure represented by the structural coordinates in Table 1.

The present invention is related to a complex of human magnesium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1 and glutathione which has a three dimensional structure represented by the structural coordinates in Table 2.

The present invention is related to a complex of human calcium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1, glutathione and 9,11-dideoxy-9 α,11α methanoepoxyprostaglandin F 2α (referred to as “U46” hereinafter) which has a three dimensional structure represented by the structural coordinates in Table 3.

U46 is represented by the formula:

and is an analog of prostaglandin H 2 (PGH 2 ) which is a substrate of PGDS.

The present invention is related to a complex of human magnesium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1, glutathione and U46 which has a three dimensional structure represented by the structural coordinates in Table 4.

The present invention is related to a complex of human calcium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1, glutathione and 9,11-dideoxy-9 α,11α epoxymetanoprostaglandin F 2α (referred to as “U44” hereinafter) which has a three dimensional structure represented by the structural coordinates in Table 5.

U44 is represented by the formula:

and is an analog of prostaglandin H 2 which is a substrate of PGDS.

The present invention is related to a complex of human magnesium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1, glutathione and U44 which has a three dimensional structure represented by the structural coordinates in Table 6.

The present invention is related to a complex of human calcium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1, glutathione and Cibacron Blue(trademark) (1-amino-4-{4-[4-chloro-6-(2-sulfo-phenylamino)-[1,3,5]triazine-2-ylmethyl]-3-sulfo-phenylamino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid) which has a three dimensional structure represented by the structural coordinates in Table 7.

Cibacron Blue™ is represented by the formula:

and is known as an inhibitor of PGDS (Thomson, A. M., Meyer, D. J. & Hayes, J. D. Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D 2 synthase, a novel class Sigma glutathione S-transferase. Biochem. J., 333, 317-325 (1998)).

The present invention is related to a complex of human magnesium type hematopoietic PGDS having an amino acid sequence of SEQ. ID NO 1, glutathione and 4-benzhydryloxy-1-{3-(1H-tetrazol-5-yl)-propyl}piperidine (referred to as “HQL-79” hereinafter) which has a three dimensional structures represented by the structural coordinates in Table 8.

HQL-79 is represented by the formula:

and is known to have anti-allergic activity (Matsushita N, Hizue M, Aritake K, Hayashi K, Takada A, Mitsui K, Hayashi M, Hirotsu I, Kimura Y, Tani T and Nakajima H, Pharmacological studies on the novel anti-allergic drug HQL-79: I. Anti-allergic and anti-asthmatic effects in various experimental models. Japanese Journal of Pharmacology, 78, 1-10 (1998)).

The present invention is further related to use of at least one structural coordinates selected from the group consisting of the structural coordinates represented in Tables 1-8 in selecting a compound for inhibiting human hematopoietic PGDS.

The present invention is related to a method for selecting an inhibitor of human hematopoietic PGDS, comprising steps of:

(a) providing at least one structural coordinates selected from the group consisting of the structural coordinates represented in Tables 1-8 which characterizes an active site of human hematopoietic PGDS;

(b) providing steric structure of a candidate compound; and

(c) fitting the candidate compound to the active site of human hematopoietic PGDS to select the inhibitor.

The amino acid residues involved in the active site of human hematopoietic PGDS are Tyr8, Phe9, Asn10, Met11, Arg12, Gly13, Arg14, Ala15, Glu16, Leu17, Trp39, Pro40, Glu41, Ile42, Lys43, Gly49, Lys50, Ile51, Pro52, Ile53, His 62, Gln63, Ser64, Leu65, Asp93, Thr94, Leu95, Asp96, Asp97, Phe98, Met99, Ser100, Cys101, Phe102, Phe103, Trp104, Ala105, Glu106, Lys107, Lys108, Gln109, Asp110, Val111, Lys112, Glu113, Gln114, Met115, Phe116, Tyr152, Trp153, Glu154, Ile155, Leu156, Ser157, Thr158, Thr159, Leu160, Leu161, Val162, Phe163, Thr197, Lys198, and Leu199.

The term “fit” means that a candidate compound is optimized in energy and configuration to the active site of human hematopoietic PGDS. A compound is selected in such a manner that the contact area of a candidate compound with human hematopoietic PGDS is maximum when the compound binds to the active site of human hematopoietic PGDS, and that the binding mode between the compound and the active site of human hematopoietic PGDS is maximum.

Preferably, it is confirmed that the thus selected inhibitor inhibits PGDS by contacting the inhibitor with hematopoietic PGDS in the presence of prostaglandin H 2 .

In addition, the inhibitor is confirmed whether it has at least one biological activity selected from the group consisting of anti-allergic activity, sleep control activity, anti-obesity activity and brain wound healing activity.

FIG. 1 shows the three-dimensional structure of a complex of human hematopoietic prostaglandin D synthase (PGDS)(calcium type) and glutathione. PGDS forms a dimer, and there is a metal ion-binding site in the center of the dimer.

FIG. 2 shows PGDS monomer structure viewed from the side of the monomer.

FIG. 3 shows a surface structure of PGDS viewed from the same direction as in FIG. 2 .

FIG. 4 is an enlarged view of surface structure of PGDS complexed with calcium, glutathione, and U46. Oxygen atom of cyclopentane ring of U46 forms hydrogen bond with glutathione

FIG. 5 shows the three-dimensional structure of PGDS complexed with calcium, glutathione and U44. The alpha chain of the substrate analogue is surrounded by Lys107, Lys112 and Lys198 of PGDS which are basic amino acid residues. The residues form salt-bridge with carboxylate of the α chain. In magnesium-type PGDS, similar three-dimensional structure is observed.

FIG. 6 shows three dimensional structure of PGDS complexed with magnesium, glutathione and U46.

(1) Human Hematopoietic Prostaglandin D Synthase

The amino acid sequence of human hematopoietic prostaglandin D synthase is known. The method for preparing it is also disclosed (Japanese Patent Kokai No. 322773/1997). Although it is possible to obtain the enzyme from human tissue, it is convenient to prepare it using recombinant techniques.

For example, an expression plasmid (pT7-7hPGDS) which comprises cDNA encoding human hematopoietic prostaglandin D synthase having amino acid sequence of SEQ. ID. NO. 1 under the control of T7 polymerase gene promoter is constructed (Japanese Patent Kokai No. 322773/1997). Escherichia coli BL21 (DE-3) is transformed with the expression plasmid using modified calcium chloride method (Inoue, et al., Gene 96:23-28 (1990)). Escherichia coli pYK1 transformed with the expression plasmid is deposited under accession number FERM BP-5489 in National Institute of Advanced Industrial Science and Technology.

Escherichia coli pYK1 is cultured in LB medium (containing 50 μg/ml of ampicillin) at 37° C. overnight. IPTG (isopropylthio-β-D-galactoside) is then added to final concentration of 0.4 mM and Escherichia coli pYK1 is further cultured for additional 4 to 6 hours.

After the completion of cultivation, the bacteria is fractured by ultrasonication and centrifuged at 13,000 rpm at 4° C. for 15 minutes to remove precipitates. Fractionation is effected with ammonium sulfate. Fraction of 40% to 60% saturation of ammonium sulfate is obtained and dialyzed against 50 mM sodium phosphate buffer. The dialyzed solution is applied to Glutathione Sephalose 4B column and purified by affinity chromatography.

(II) Crystal of Human Hematopoietic Prostaglandin D Synthase Complex

Crystallization of a protein utilizes depositing a protein under specific conditions as a crystal when the protein is switched from dissolving conditions to non-dissolving conditions by adding a precipitating agent to a solution of the protein of interest, or by reducing the amount of solution by vaporizing solvent.

The inventors have found that human hematopoietic PGDS complex is successfully crystallized with the hanging drop vapor diffusion method. The hanging drop vapor diffusion method is a method wherein a mixed solution of protein solution with a precipitating solution is hanged up on a glass plate using surface tension and the solution is enclosed with another precipitating solution having a higher concentration in a sealed space to form a crystal of the protein. Due to vapor diffusion, the concentration of the precipitating agent is gradually increased to obtain a crystal. When a large crystal is required, a crystal obtained in a first crystallization and having a size of about 0.01 mm is selected and re-crystallized again with a macro-seeding method (Stura, E. A. & Wilson, L. A. Applications of the streak seeding technique in protein crystallization. J. of Crystal Growth, 110, 270-282 (1991).) to obtain a crystal having size of about 0.3×0.3×1.5 mm, which is proper for X-ray diffraction experiments.

The conditions for preparing eight human hematopoietic PGDS complexes are described hereinafter.

(1) Complex of human hematopoietic PGDS with calcium and Glutathione

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM  100 mM  Admixture Glutathione 5 mM 10 mM dithiothreitol 5 mM 10 mM Calcium 2.5 mM    5 mM chloride Dioxane  1%  2% Protein PGDS   5 mg/mL — Temperature: 20° C., pH: 8.4

(2) Complex of human hematopoietic PGDS with magnesium and glutathione

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM  100 mM  Admixture Glutathione 5 mM 10 mM dithiothreitol 5 mM 10 mM Magnesium 2.5 mM    5 mM chloride Dioxane  1%  2% Protein PGDS   5 mg/mL — Temperature: 20° C., pH: 8.4

(3) Complex of human hematopoietic PGDS with calcium, glutathione and 9,11-dideoxy-9α,11α-methanoepoxyprostaglandine F 2α (U46)

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM  100 mM  Admixture Glutathione 5 mM 10 mM dithiothreitol 5 mM 10 mM Calcium 2.5 mM    5 mM chloride Dioxane  1%  2% U46 10 mM  — Protein PGDS   5 mg/mL — Temperature: 20° C., pH: 8.4

(4) Complex of human hematopoietic PGDS with magnesium, glutathione and 9,11-dideoxy-9α,11α-methanoepoxyprostaglandine F 2α (U46)

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM 100 mM  Admixture Glutathione  5 mM 10 mM dithiothreitol  5 mM 10 mM Magnesium 2.5 mM   5 mM chloride Dioxane  1%  2% U46 10 mM — Protein PGDS    5 mg/mL — Temperature: 20° C., pH: 8.4

(5) Complex of human hematopoietic PGDS with calcium, Glutathione and 9,11-dideoxy-9α,11α-epoxymethanoprostaglandine F 2α (U44)

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM  100 mM  Admixture Glutathione 5 mM 10 mM dithiothreitol 5 mM 10 mM Calcium 2.5 mM    5 mM chloride Dioxane  1%  2% U46 5 mM — Protein PGDS   5 mg/mL — Temperature: 20° C., pH: 8.4

(6) Complex of human hematopoietic PGDS with magnesium, glutathione and 9,11-dideoxy-9α,11α-epoxymethanoprostaglandine F 2α (U44)

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM  100 mM  Admixture Glutathione 5 mM 10 mM dithiothreitol 5 mM 10 mM Magnecium 2.5 mM    5 mM chloride Dioxane  1%  2% U44 5 mM — Protein PGDS   5 mg/mL — Temperature: 20° C., pH: 8.4

(7) Complex of human hematopoietic PGDS with calcium, glutathione and Cibacron Blue (trademark) (1-amino-4-{4-[4-chloro-6-(2-sulfo-phenylamino)-[1,3,5]triazine-2-ylmethyl]-3-sulfo-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulfonic acid)

Composition:

Internal Sol. External Sol. Precipitating PEG 6000 14% 16% agent Buffer Tris buffer 50 mM  100 mM  Admixture Glutathione 5 mM 10 mM dithiothreitol 5 mM 10 mM Calcium 2.5 mM    5 mM chloride Dioxane  1%  2% Cibacron 1 mM — Blue Protein PGDS   5 mg/mL — Temperature: 20° C., pH: 8.4

(8) Complex of human hematopoietic PGDS with calcium, glutathione and 4-benzhydryloxy-1-{3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79)

Composition:

(III) Three-Dimensional Coordinate of Human Hematopoietic PGDS Complex

Three-dimensional structure of human hematopoietic PGDS complex was clarified using X-ray crystallography from the crystal of human hematopoietic PGDS complex obtained from (II).

Data collection was carried out for a mercury derivative crystal of human hematopoietic PGDS of the calcium type in SPring-8 RIKEN beam line BL45XD in consideration of the anomalous dispersion effect. Data collection was carried out using synchrotron radiation lights of three wavelengths of 1.009 Å, 1.04 Å and 1.10 Å to calculate eight positions of mercury from the analysis of the Patterson function. The position was precisely determined using the program MLPHARE (Otwinowski, Z. in Proceedings of CCP4 Study Weekend, Isomorphous Replacement and Anomalous Scattering, edited by Sawyer, L., Issacs, N., and Bailey, S. [Science and Engineering Research Council (England) Daresbury Lab., Warrington, U.K.], pp. 80-86 (1993)) to obtain electron density of the enzyme. An enzyme model was constructed on a graphics workstation to obtain a highly accurate enzyme model of human hematopoietic PGDS of the calcium type using the refinement program CNS (Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Cryst., D54, 905-921 (1998)).

The other structural model was obtained in high accuracy with the program CNS using the calcium type model firstly obtained as the search model.

Crystallographic data obtained for the crystals prepared in (II)(1) to (II)(8) are as follows:

Table 1 represents the structural coordinates of the complex of human hematopoietic PGDS with calcium and glutathione; Table 2 represents the structural coordinates of the complex of human hematopoietic PGDS with magnesium and glutathione; Table 3 represents the structural coordinates of the complex of human hematopoietic PGDS with calcium, glutathione and 9,11-dideoxy-9α,11α-methanoepoxyprostaglandine F 2α (U46); Table 4 represents the structural coordinates of the complex of human hematopoietic PGDS with magnesium, glutathione and 9,11-dideoxy-9α,11α-methanoepoxyprostaglandine F 2α (U46); Table 5 represents the structural coordinates of the complex of human hematopoietic PGDS with calcium, glutathione and 9,11-dideoxy-9α,11α-epoxymethanoprostaglandine F 2α (U44); Table 6 represents the structural coordinates of the complex of human hematopoietic PGDS with calcium, glutathione and 9,11-dideoxy-9α,11α-epoxymethanoprostaglandine F 2α (U44); Table 7 represents the structural coordinates of the complex of human hematopoietic PGDS with calcium, glutathione and Cibacron Blue (trademark); Table 8 represents the structural coordinates of the complex of human hematopoietic PGDS with magnesium, glutathione and 4-benzhydryloxy-1-{3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79).

Each table represents three-dimensional structural coordinates according to the format of Protein Data Bank (rcsb. org/pdb/, USA). “ATOM” at the first column indicates that the row describes atom coordinates; the second column indicates the atom number; the third column indicates the atom type in the amino acid residue or the like, for example, carbonyl carbon atom is represented by C, and carbonyl oxygen atom is represented by O; the fourth column indicates the amino acid residue or the like; the fifth column indicates the class of molecule; the sixth column indicates the amino acid number; the seventh, eighth, and ninth columns indicate coordinates of the atom (in Å for X-axis, Y-axis, and Z-axis directions in the order); the tenth column indicates the occupancy of the atom (in the present invention 1.00 for all atoms); and the eleventh column indicates the temperature factor of the atom. The twelfth column indicates the class of molecule like the fifth column.

(IV) Three-Dimensional Structure and Binding Site of Human Hematopoietic PGDS Complex

(1) Complex of Human Hematopoietic PGDS with Calcium and Glutathione

Prostaglandin D synthase form a dimer and calcium ion exists in the center of the dimer (see FIG. 1 ). Calcium has no influence on the glutathione binding, but increases the reaction rate of PGDS enzyme. Glutathione binds to Tyr8, Arg14, Trp39, Gly49, Lys50, Ile51, Pro52, Gln63 and Ser64 of PGDS.

(2) Complex of Human Hematopoietic PGDS with Magnesium and Glutathione

In accordance with the change of metal ion in the center of the dimer from calcium ion to magnesium ion, coordination structure of water molecule to the metal ion is significantly changed. Magnesium increases the affinity of glutathione to PGDS by more than 3 times.

(3) Complex of Human Hematopoietic PGDS with Calcium, Glutathione and 9,11-dideoxy-9α,11α-methanoepoxyprostaglandine F 2α (U46)

In the complex, there exists calcium ion in the center of the dimer as in (1). In addition, substrate analog, U46, binds to PGDS. U46 binds to Tyr8, Gly3, Arg14, Gln36, Trp104, Gln36, Trp104, Gln109, Lys112, Tyr152, Lys198, and Leu199 of PGDS, and GSH. Carboxyl group of U46 is hydrogen bonded to either of Gln109, Lys112, or Lys198 of PGDS. Oxygen atom at C11 site of U46 is positioned at 4.6 Å from S atom of GSH. The ω chain of U46 approaches Tyr152 existing in the deepest portion of substrate binding site to van der Waals contact distance.

(4) Complex of Human Hematopoietic PGDS with Magnesium, Glutathione and 9,11-dideoxy-9α,11α-methanoepoxyprostaglandine F 2α (U46)

The structure of the complex differs from that of (3) in that magnesium ion exists in the center of the dimer. Although the α chain of U46 is positioned near Gln109, Lys112 and Lys198 of PGDS, it is not hydrogen bonded but is in van der Waals contact. Oxygen atom at 11 site of U46 is hydrogen bonded to Gln36 of PGDS through water molecule. The OH group in the ω chain of U46 is hydrogen bonded to rotated Arg14 and the ω chain is out of the pocket. Ether which is a solvent for U46 binds at the aperture between Trp104 of PGDS and U46 molecule.

(5) Complex of Human Hematopoietic PGDS with Calcium, glutathione and 9,11-dideoxy-9α,11α-epoxymethanoprostaglandine F 2α (U44)

The complex differs from that of (1) to (4) in that a substrate analog U44 binds to PGDS. In the calcium-binding structure, although the binding site of U44 is the same as that of U46, the manner of hydrogen bonding differs. Although they are both in the calcium-binding form, being different from U46, α-chain of U44 is hydrogen bonded with to all of Gln109, Lys112, and Lys198 of PGDS, and oxygen atom at 9 site is hydrogen bonded through water to Gln36. The α-chain of U44 is about 5 Å apart from back Tyr152, differing from the structure of (3).

(6) Complex of Human Hematopoietic PGDS with Magnesium, Glutathione and 9,11-dideoxy-9α,11α-epoxymethanoprostaglandine F 2α (U44)

Although this complex is identical with (5) in that they are complexes with substrate analog U44, it is different from (5) in that the metal ion positioned in the center of dimer is magnesium ion.

(7) Complex of Human Hematopoietic PGDS with Calcium, Glutathione and Cibacron Blue (Trade Mark)

The complex differs from the above six structures in that it is a complex with an inhibitor Cibacron Blue. Metal ion in the center of the dimer is calcium. Cibacron Blue binds to Phe9, Gly10, Gly13, Arg14, Gln36, Met99, Ser100, Trp104, Glu106, Lys107, Gln109, Lys112, Tyr152, Cys156, Lys198, and Leu199 of PGDS, and GSH

(8) Complex of Human Hematopoietic PGDS with Magnesium, Glutathione and 4-benzhydryloxy-1-{3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79)

The complex is different from (1) to (6) in that it is a complex with an inhibitor HQL-79, and an inhibitor different from (7) is used. Magnesium ion is bonded in the center of the dimer. Induce-fit phenomenon is caused due to the binding of HQL-79, and space group is changed from P2 1 to P1 1 . HQL-79 binds to Tyr8, Phe9, Arg14, Met99, Phe102, Trp104, Tyr152, Ile155, Thr159, Phe163, and Leu199 of PGDS and GSH.

(V) Use of Three Dimensional Coordinates for Preparing Inhibitor of Human Hematopoietic Prostaglandin D Synthase

A compound capable of inhibiting human hematopoietic PGDS can be selected using three-dimensional coordinates of Tables 1 to 8.

The present invention is related to a method for selecting an inhibitor of human hematopoietic PGDS, comprising steps of:

(a) providing at least one of these structural coordinates selected from the group consisting of the structural coordinates represented in Tables 1-8 which characterizes an active site feature of human hematopoietic PGDS;

(b) providing steric structure of a candidate compound; and

(c) fitting the candidate compound to the active site of human hematopoietic PGDS to select the inhibitor.

The amino acid residues involved in the active site of human hematopoietic PGDS are Tyr8, Phe9, Asn10, Met11, Arg12, Gly13, Arg14, Ala15, Glu16, Leu17, Trp39, Pro40, Glu41, Ile42, Lys43, Gly49, Lys50, Ile51, Pro52, Ile53, His 62, Gln63, Ser64, Leu65, Asp93, Thr94, Leu95, Asp96, Asp97, Phe98, Met99, Ser100, Cys101, Phe102, Phe103, Trp104, Ala105, Glu106, Lys107, Lys108, Gln109, Asp110, Val111, Lys112, Glu113, Gln114, Met115, Phe116, Tyr152, Trp153, Glu154, Ile155, Leu156, Ser157, Thr158, Thr159, Leu160, Leu161, Val162, Phe163, Thr197, Lys198, and Leu19

Based on the three-dimensional structure information, commercially available compounds are one by one minimized in energy using various programs so that the binding to the active site of the enzyme occurs in the most appropriate orientation. This procedure is automatically effected for all commercially available compounds to compare free energy to select compounds which readily bind to the active site of the enzyme. A compound thus obtained is used as basic backbone, and more stable derivative is designed to synthesize more stable compound in order to develop a new inhibitor of the enzyme.

It is preferred to design inhibitors using a computer. For example, Indigo 2, a workstation supplied by Silicon Graphics, Inc., is suitable as a computer used for designing inhibitors. However, the computer is not limited to this one, and any computer may be used so long as it is capable of running an appropriate program. Likewise, there is no particular limitation on the computer storage medium. For example, Insight II, a computer program commercially available from Accelrys, Inc. may be used as a program for designing. In particular, the programs Ludi or DOCK, a module of Insight II specially prepared for such purposes, may be used alone or in combination to facilitate identification, searching, evaluation, or designing.

In designing of inhibitor, there are conceptually two steps. The first step is to find a compound which serves as a starting point for drug design, known for those skilled in the art as a lead compound. The next step is optimization of the lead compound wherein compounds having better properties as medicines, for example, having better activity, having better pharmacokinetics, or having less toxicities and side effects are sought starting from the lead compound.

The step in which a lead compound is found using the structure coordinates of the PGDS complex provided by the present invention is achieved, for example, using a database in a computer into which structures of plural compounds have been entered, by a method in which interactions between three-dimensional structures of a compound in the database and PGDS are sorted out in a visual manner one after another, or by a method in which amplitudes of binding energy are calculated one after another using a computer and compounds which stably bind to PGDS are found from the database. Although it is preferred that the database of compound's structures contains determined three-dimensional structure coordinates entered therein, for low molecular weight compounds, it does not have to be a database of three-dimensional structure coordinates, because such low molecular weight compounds may change their conformations relatively freely, and also because three-dimensional structure coordinates for each conformation can be derived by calculations in a relatively short time. In the latter cases, information for chemical covalent bonds of low molecular weight compounds are entered into the database.

Specifically, in the visual method, PGDS complexes are firstly displayed on a computer screen according to the structure coordinates of the present invention. In this step, although a three-dimensional representation may be made on the computer screen using, for example, Crystal Eye as described above, visual examinations can also be achieved without using such a three-dimensional representation.

Chemical interactions to be considered include electrostatic interactions, hydrophobic interactions, hydrogen bonding, van der Waals interactions, and the like. Thus, the structure should be comprehensively examined whether it is favorable for interactions, for example, so that functional groups which tend to bear negative charges such as carboxyl groups, nitro groups, and halogens interact with amino acid residues in PGDS having positive charge such as lysine, arginine, and histidine, so that functional groups which tend to bear positive charge such as amino, imino, and guanidyl groups interact with amino acid residues in PGDS having negative charge such as glutamic acid and aspartic acid, so that hydrophobic functional groups such as aliphatic groups and aromatic groups interact with hydrophobic amino acid residues such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine, so that functional groups involved in hydrogen bonding such as hydroxyl and amide groups can form hydrogen bonds with backbone or side chain portions of PGDS, so that binding between the compound and PGDS causes no steric hindrance, and so that empty spaces are filled to minimize such empty spaces and maximize van der Waals interactions. Thus, electrostatic interactions, hydrophobic interactions, van der Waals interactions, hydrogen bonding, and other factors are visually and comprehensively considered to finally determine whether or not the compound is suitable as a lead compound.

In the method of energy evaluation with a computer, the energy of binding between a compound and PGDS is determined by molecular force field calculations. Such calculations are applied to each compound in the database to find compounds which may serve as a lead compound capable of stable binding. As a molecular force field used in the calculations, for example, CVFF, AMBER force field optimized for proteins, which is contained in DISCOVER module of Insight II program may be used. In addition, some computer programs like Ludi in Insight II can automatically output candidates for lead compound when three-dimensional structure coordinates of interacting amino acid residues in a protein molecule are given, and such programs may also be applied to the method of present invention.

Furthermore, the visual examinations and the examination considering energy are not strictly sorted out from each other, and both techniques may be used in combination as appropriate.

The next step, in which optimization of the lead compound is conducted using the structure coordinates of the PGDS complex is used for the purpose of, where a lead compound which binds to PGDS has already been found by the above method or separately found in an experimental manner, optimizing the lead compound to obtain a better compound such as a compound having higher biological activities as an inhibitor or a compound having a structure favorable for oral administration as a medicine. It becomes possible only after a precise picture of chemical bonding between the lead compound and PGDS has been elucidated to directly find a site which is not optimal for interactions between the lead compound and PGDS and to design a new compound having an optimal functional group at that site, thereby enabling to design a more optimized compound.

For visual examinations with a computer, a model of the complex between the lead compound and PGDS is firstly displayed on a computer screen by entering the three-dimensional structure coordinates of the lead compound and the structure coordinates of PGDS provided by the present invention into a computer on which a computer program expressing three-dimensional coordinates of molecules runs or into a storage medium of the computer. In this step, although a three-dimensional representation may be made on the computer screen using, for example, Crystal Eye as described above, visual examinations can also be achieved without using such a three-dimensional representation. It is a logical to design a compound to modify the lead compound so as to yield a compound more favorably interacting with PGDS or a compound having better pharmacokinetics while retaining such interactions.

Chemical interactions to be considered are the same as those in the step to find a lead compound, and a new compound having better properties as an inhibitor is finally designed starting from the lead compound.

In the method by energy evaluation with a computer, the energy of binding between a new compound designed from the lead compound and PGDS is determined by molecular force field calculations to judge the validity of the design. In addition, it is also possible to use a method in which other molecules such as solvent molecules are additionally included in the model and the free energy is determined using molecular dynamics to derive a compound capable of stable binding. As a molecular force field used in the calculations, for example, CVFF, AMBER force field optimized for proteins, which is contained in DISCOVER module of Insight II program may be used.

Furthermore, the visual examinations and the method by energy evaluations may be used in combination as appropriate.

After a candidate compound for inhibition of hematopoietic PGDS is thus selected, the selected compound is contacted with the enzyme in the presence of the substrate (prostaglandin H 2 ) to confirm the compound can inhibit the enzymatic activity (Shimizu, T., Yamamoto, S., and Hayaishi, O. (1979). Purification and properties of prostaglandin D synthase from rat brain. J. Biol. Chem. 254, 5222-5228). The measurement of enzymatic activity is, for example, carried out as follows: The substrate [1- 14 C]prostaglandin(PG)H 2 is prepared by reacting [1- 14 C]arachidonic acid with cyclooxygenase. Since PGH 2 is easily decomposed in aqueous solution (half-life: about 5 minutes), it is dried and stored at a low temperature (−80° C.). The enzymatic reaction is carried out by injecting with microsyringe 1 μl of PGH 2 solution (acetone or nonvolatile diethleneglycohol solution) to 50 μl of 0.1 M phosphate buffer (pH 7.5) containing 1 mM glutathione and the enzyme. After the reaction is effected for 30˜60 seconds, the reaction is quenched by adding 300 μl of ice-cooled ether/methanol/0.1M citric acid mixture (20:4:1 v/v/v), and the substrate and the reaction product are extracted with ether under acidic condition. Anhydrous sodium sulfate is then added to the reaction solution to remove water. An aliquot (about 50 μl) of organic layer is applied on silica gel thin layer in a cool room (about 4° C.) and silica gel thin layer chromatography (development solvent: ether/methanol/acetic acid (9:2:0.1)) is carried out in a freezer (−20° C.). After the development, radioactivity of PGD 2 fraction and other fraction are measured and the enzyme activity is calculated from the ratio of conversion to PGD 2 .

Alternatively, enzyme reaction is carried out using commercially available non-labeled PGH 2 . After the reaction, PGH 2 is decomposed to 12(S)-hydroxy-8,10-trans-5-cis-heptadecatrienolic acid with the treatment by FeCl 2 before quantifying with reverse phase HPLC using 11-β-PGE 2 as an internal standard or commercially available ELISA to quantify PGD 2 .

Since an inhibitor of hematopoietic PGDS can be an anti-allergic agent, a sleep control agent, an anti-obesity agent, and brain wound healing agent, pharmacological activity of the inhibitor is measured as each medicine.

A method for measuring pharmacological activity as an anti-allergic agent is described in Fugner A, Bechtel W. D., Kuhn, F. J. and Mierau, J. In vitro and in vivo studies of the non-sedating antihistamine epinastine. Arzneimittelforschung, 38, 1446-1453 (1988); Kamei, C., Izushi, K., Adachi, Y., Shimazawa, M. and Tasaka, K. Inhibitory effect of epinastine on the type II-IV allergic reactions in mice, rats and guinea pigs. Arzneimittelforschung, 41, 1150-1153 (1991). Activity of an anti-allergic agent is measured as follows: Candidate prostaglandin D synthase inhibitors are administered to an animal or cells. Allergic reactions in animals or the production of prostaglandin D 2 from cells is then measured to evaluate the activity of the candidate compound. Alternatively, for example, any allergen is administered to animal before a candidate compound for prostaglandin D synthase inhibitor is administered and systemic allergic reaction of the animal is observed to evaluate efficacy of the candidate compound. Alternatively, for example, any allergen is administered to an animal to put the animal in an allergic condition. Cells are collected from the animal, and the candidate compound of prostaglandin D synthase inhibitor is added to the cells in vitro. The amount of prostaglandin D 2 caused by the stimulation of the cells with allergen is measured to evaluate of efficacy of the candidate compound.

A method for measuring pharmacological activity as sleep control agent is described in Huang, Z.-H., Qu, W.-M., Li, W.-D., Mochizuki, T., Eguchi, N., Watanabe, T., Urade, Y. & Hayaishi, O. Arousal effect f orexin A depends on activation of the histaminergic system. Proc. Natl. Acad. Sci. USA, 98, 9965-9970 (2001). For example, the activity is measured as follows: When prostaglandin D synthase inhibitor is administered in ventricle of the brain of a mouse, significant sleep disturbance occurs. On the other hand, when prostaglandin D 2 is administered in the ventricle of the brain of a mouse, sleep is induced. Accordingly, the candidate compound of prostaglandin D synthase inhibitor is administered to an animal and the sleep condition is observed to evaluate the medical efficacy of the candidate compound. The measurement of the sleep condition of animal is, for example, carried out by measuring brain wave, electromyogram, activity, feeding and water-drinking amount, temperature, etc. from time to time.

A method for measuring pharmacological activity as anti-obesity agent is described in Ikeda H, Taketomi S, Sugiyama Y, Shimura Y, Sohda T, Meguro K & Fujita T. Effects of pioglitazone on glucose and lipid metabolism in normal and insulin resistant animals. Arzneimittelforschung, 40, 156-162 (1990) and Sohda T, Mizuno K, Momose Y, Ikeda H, Fujita T & Meguro K. Studies on anti-diabetic agents. 11. Novel thiazolidinedione derivatives as potent hypoglycemic and hypolipidemic agents. J. Med. Chem., 35, 2617-2626 (1992).

The measurement of pharmacological activity as anti-obesity agent is carried out as follows: The Prostaglandin D synthase inhibitor candidate is administered to an animal and the activity of the candidate compound is measured by measuring the obesity of the animal. For example, high fatty food is given to an animal and the candidate compound of prostaglandin D synthase inhibitor is then administered to the animal. The Obesity condition of animal is, for example, estimated by measuring body weight, active mass, feeding amount, fat weight, biochemical value in blood, etc. from time to time.

The measurement of activity of brain wound healing agent in individual is carried out as follows: Medical efficacy of the candidate compound is measured by administering the candidate compound of prostaglandin D synthase inhibitor to an animal and then measuring the degree of brain wound healing. For example, it is measured by administering the candidate compound of prostaglandin D synthase inhibitor to a traumatic cerebral cortex wound (Stab wound) model (Salhia B et al, Brain Res., 888:87-97, 2000; Asahi M., et al., J. Neurosci., 21:7724-7732, 2001; Garcia de Yebenes E., et al., J. Neurochem., 73:812-1999). The measurement of brain wound healing is effected by measuring for example, amount of behavior, immunohistochemical staining, amount of expressed gene.