WO2006025425A1

WO2006025425A1 - Protein participating in inhibition of plasmodium growth

Info

Publication number: WO2006025425A1
Application number: PCT/JP2005/015855
Authority: WO
Inventors: Masataka Ihara; Kiyosei Takasu
Original assignee: Japan Science And Technology Agency; National University Corporation Tohoku University
Priority date: 2004-09-01
Filing date: 2005-08-31
Publication date: 2006-03-09
Also published as: JP2006067867A

Abstract

It is intended to provide a protein having a high affinity for rhodacyanine, which is a drug inhibiting the growth of malarial parasite (Plasmodium sp.) being parasitic on (infecting) erythrocytes, (namely, a protein participating in the inhibition of Plasmodium growth); a method of isolating the protein; a gene encoding the protein participating in the inhibition of Plasmodium growth as described above; and a method of screening an antimalarial remedy and a preventive targeting the protein participating in the inhibition of Plasmodium growth as described above. An affinity gel having rhodacyanine dye as a ligand is prepared and a protein mixture originating in Plasmodium sp. is adsorbed by the affinity gel. Then the target protein closely relating to the expression of an antimalarial activity is separated by taking advantage of the difference in affinities of the proteins for the affinity gels.

Description

Proteins involved in malaria growth inhibition

Technical field

[0001] The present invention relates to a protein (that is, a protein involved in malaria growth inhibition) having a high affinity for oral dacyanin, which is a drug that inhibits the growth of Plasmodium sp. The present invention relates to an isolation method, a gene encoding a protein involved in malaria growth inhibition, and a screening method for an antimalarial therapeutic agent and preventive agent targeting the protein involved in malaria growth inhibition.

Background art

[0002] Malaria mediated by anopheles mosquitoes continues to cause tremendous damage in ancient power. 2 According to a WHO report in 002, there are currently about 300 million malaria infected persons, of which 1.1 million are estimated to die each year, mainly children. However, malaria suffering is not evenly distributed throughout the world. For example, in Japan after the end of the war in 1946, it was almost completely suppressed by eradication projects such as DDT spraying for the control of power-borne mosquitoes, which were widespread. This suppression is influenced quite closely by the fact that Japan has winter. This is because the growth of plasmodium in mosquitoes (sexual reproduction) does not occur below 16 ° C, and the life cycle is thought to be almost stopped. On the other hand, in the tropical and subtropical regions without winter, the same eradication movement is inevitable, and malaria is still raging. In fact, 90% of the deaths are African. Under these circumstances, people living in temperate zones should not be relieved easily. Recent deforestation, long-term weather changes due to the El-Io-yo phenomenon and global warming have brought the mosquito habitat northward. In addition, considering the increase in imported malaria brought into Japan by Japanese travelers and foreigners visiting Japan, malaria is a disease that can never be neglected even in temperate regions including Japan.

[0003] There are about 200 types of malaria parasites that parasitize primates, birds, reptiles, etc., but human parasitic species include Plasmodium falciparum, Plasmodium falciparum ( P. vivax), P. malariae (P. malariae), oval malaria (P. ova) 4) are typical. Among them, P. falciparum malaria has the risk of severe cerebral malaria, so the development of an anti-P. Falciparum drug that has a high mortality rate is most necessary.

[0004] The life cycle of malaria is described below (for example, see Non-Patent Document 1). First, when an mosquito sucks blood, infectious insects (sporozoites) gathered in the salivary glands of the mosquito enter the human blood. The protozoa that enter the blood quickly migrate to the liver, incubate for 1-2 weeks, and repeat proliferation in hepatocytes to form merozoites, daughter bodies. Eventually this destroys hepatocytes and enters the red blood cells. Part of the reproduction cycle in erythrocytes is differentiated into male and female reproductive maternal (gametocytes). The life cycle is repeated by sucking blood of this gametite by the spotted mosquito. The protozoa sexually reproduce in the body of mosquitoes, but repeats a large number of asexual reproductions in the human body.

[0005] Conventionally, quinine, black mouth quince, artemisinin, pyrimesamine, and atobacon have been used as a therapeutic or preventive agent for malaria. Any preventive agent cannot guarantee protection from infection. The present inventors aimed to develop a novel antimalarial agent, and found in the process that the compound MKT-077 having an oral dasianin skeleton exhibited an antimalarial activity comparable to black mouth quinone in vitro (EC = 70 nM, selectivi

50

ty = 210). MKT-077 is a compound classified as an oral dasyanin pigment because it has both a cyanine structure and a merocyanine structure with the oral danin ring as the mother nucleus. As a result of derivative synthesis, we found more effective MKH-57 (EC = 12 nM, selectivity = 1000)

50

For example, see Patent Documents 1 to 4). Also reported is the synthesis of oral dasyanin by combinatorial synthesis and the antimalarial activity of the synthesized compound (see, for example, Patent Document 5).

[0006] Further, the present inventors have clarified that MKT-077 is accumulated at a specific site in P. falciparum by observation using a fluorescence microscope (for example, Patent Document 6). reference).

Patent Document 1: Japanese Patent Laid-Open No. 2000-191531

Patent Document 2: JP 2003-034640 A

Patent Document 3: Japanese Patent Application Laid-Open No. 2003-034641

Patent Document 4: Japanese Patent Laid-Open No. 2003-034642 Patent Document 5: Japanese Unexamined Patent Application Publication No. 2004-137271

Patent Document 6: Japanese Unexamined Patent Application Publication No. 2004-144526

Non-patent literature l: Nature 2002, 419, 495-496

Disclosure of the invention

Problems to be solved by the invention

[0007] Forces that are known to have anti-malarial activity of oral dassaninhi compounds It has been unknown which component of the malaria parasite interacts with the anti-malarial activity. In the development of pharmaceuticals, it is important to clarify biomolecules such as proteins that cause disease. It is possible to design organic compounds as pharmaceuticals with computer support based on the information of the three-dimensional structure of the protein. In addition, by identifying the gene encoding the corresponding protein, it becomes possible to develop therapeutic agents and preventive vaccines at the gene level. An object of the present invention is to provide a method for isolating a protein derived from a malaria parasite having an affinity for a rhodacinian compound, and a protein expressing antimalarial activity obtained by such an isolation method. Means for solving the problem

[0008] As described above, since there was a good correlation between the specific accumulation phenomenon of oral dasyanin and high antimalarial activity, it was closely related to the expression of antimalarial activity of oral dasyanin pigment at the site of pigment accumulation. Presumed that the target protein was present. Therefore, we decided to prepare a affili-gel with a mouth dashianine dye as a ligand and perform affinity-take mouth-matography to isolate the target protein. Affinity-take-matography is a method in which a protein solution is allowed to flow continuously through a column packed with a chromatographic support on which various ligands are immobilized, and the target protein is bound to the ligand. Is dissociated from the ligand and collected. By this method, the protein mixture derived from Plasmodium is adsorbed on the affili-gel and fractionated into fractions using the difference in affinity of each protein, and the fractions separated by SDS-polyacrylamide gel electrophoresis. The present inventors have found a protein that expresses antimalarial activity of approximately 27, 29, 31, 33, and 37 kilodaltons, and have completed the present invention.

[0009] That is, the present invention is derived from [1] Plasmodium sp. An isolated protein that exhibits antimalarial activity by affinity with a compound represented by formula (1)

[Chemical 2]

(In the formula, R \ R ² and R ° each represent a hydrogen atom, an unsubstituted or substituted alkyl group, an alkyl group, an aryl group, or a heterocyclic group, which may be the same or different. A and B represent a nitrogen-containing heterocyclic ring, n represents an integer of 0, 1 or 2, and represents a pharmaceutically acceptable cation.

The present invention also provides [2] the isolated protein of claim 1 having a partial amino acid sequence of TIDNNNIDEK, and [3] a size determined by SDS-polyacrylamide gel electrophoresis is approximately 33 kilodaltons. The isolated protein according to [2] above, [4] a gene encoding the isolated protein according to [2] or [3] above, [5] the partial amino acid sequence of CNN NNNNNEK [ 1) the isolated protein according to [6], the size determined by SDS-polyacrylamide gel electrophoresis is approximately 27 kilodaltons, or the protein according to [5] above, [7] A gene encoding the isolated protein according to [5] or [6], or [8] a single protein according to [1], wherein the size determined by SDS-polyacrylamide gel electrophoresis is about 29 kilodaltons. Released tamper Quality and, [9] SDS-size determined by polyacrylamide gel electrophoresis [1] proteins and isolated according is approximately 31 kg Dalton, [10] SDS—The isolated protein according to [1] above, which has a size determined by polyacrylamide gel electrophoresis of about 37 kilodaltons.

Furthermore, the present invention is [11] a method for isolating a protein derived from a Plasmodium sp. Parasitic on erythrocytes and expressing an antimalarial activity by affinity with a compound represented by the formula (1). Thus, the affiliation skeleton is used as a ligand site, and a affinity gel is produced by covalently bonding to the agarose gel. The protein mixture derived from protozoan parasite is adsorbed to the powerful affinity gel, and the affinity between the affinity gel and each protein is obtained. A protein that expresses antimalarial activity, characterized by separating the protein that expresses antimalarial activity by SDS-polyacrylamide gel electrophoresis. And [12] a protein that expresses antimalarial activity, TIDNNNIDEK, which has a partial amino acid sequence of [11] A method for isolating a protein that expresses rear activity, or [13] The protein that expresses antimalarial activity has a size determined by SDS-polyacrylamide gel electrophoresis of about 33 kilodaltons. A method for isolating a protein that expresses antimalarial activity, [14] a method for isolating a protein that expresses antimalarial activity according to [11] above, which has a partial amino acid sequence of CNNNNNNNEK, a protein that expresses antimalarial activity, [15] Protein strength that expresses antimalarial activity The method for isolating a protein that expresses antimalarial activity according to [14] above, wherein the size determined by SDS-polyacrylamide gel electrophoresis is about 27 kilodaltons. [16] Proteins that express antimalarial activity can be of a size determined by SDS-polyacrylamide gel electrophoresis. The method for isolating a protein that expresses antimalarial activity as described in [11] above, which is 29 kilodaltons, or [17] a protein that expresses antimalarial activity is approximately the size determined by SDS-polyacrylamide gel electrophoresis. The method of isolating a protein that expresses antimalarial activity as described in [11] above, which is 31 kilodaltons, and [18] a protein that expresses antimalarial activity is approximately the size determined by SDS-polyacrylamide gel electrophoresis. The method for isolating a protein expressing antimalarial activity according to [11] above, which is 37 kilodaltons, [19] above [1] to [3], [5], [6], [8] to [ The isolated protein according to any one of [10] is brought into contact with the test substance, and the affinity between the isolated protein and the test substance is increased. The present invention relates to a screening method for a substance having an antimalarial activity, characterized by measuring and evaluating the degree.

Brief Description of Drawings

[0013] [Fig. 1] The present invention uses a mouth dashianine skeleton as a ligand site to produce a affiliation gel covalently linked to an agarose gel, and adsorbs a malaria parasite-derived protein mixture to the powerful affinity gel. FIG. 2 is a flow diagram showing an outline of a method for separating malaria parasite protein.

[Figure 2] Affinity-taken with the mouth dasyanin skeleton as a ligand site, covalently bonded to agarose gel, and adsorbing the malaria parasite-derived protein mixture to the powerful affinity gel FIG. 6 is a flowchart showing an outline of an experimental operation of mouth matography.

FIG. 3 is a diagram showing the results of SDS-P AGE of a protein derived from a malaria parasite isolated by the affinity gel represented by formula (3).

FIG. 4 shows the results of SDS-PAGE of a protein derived from a malaria parasite isolated by the affinity gel represented by formulas (4) to (6).

FIG. 5 is a diagram showing the results of SDS-P AGE of a malaria parasite-derived protein obtained by affine-take mouth chromatography (gradient column method) using the affili-gel represented by formula (3).

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The isolated protein of the present invention is not particularly limited as long as it is derived from a malaria parasite parasitized in erythrocytes and exhibits antimalarial activity by affinity with the compound represented by the formula (1). A protein with a partial amino acid sequence of TIDNNNIDEK (SEQ ID NO: 1), such as a protein with a size of approximately 33 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis, or a partial amino acid of CNNNNNNNEK (SEQ ID NO: 2) Proteins with sequences, such as proteins with a size determined by SDS-polyacrylamide gel electrophoresis of approximately 27 kilodaltons, proteins with a size determined by SDS-polyacrylamide gel electrophoresis of approximately 29 kilodaltons, etc. SDS-polyacrylamide gel electrophoresis determines the size. Other 31 kg Specific examples include proteins that are daltons and proteins that are approximately 37 kilodaltons in size as determined by SDS-polyacrylamide gel electrophoresis. These proteins of the present invention can be obtained by the method for isolating a protein expressing the antimalarial activity of the present invention described later.

[0015] [Chemical 3]

[0016] The present invention also relates to a gene encoding the isolated protein of the present invention.

The method for obtaining and preparing the gene of the present invention is not particularly limited, and the amino acid sequence determined by measuring the two-dimensional mass spectrum (MS-MS) of the isolated protein of the present invention. An appropriate gene can be isolated by preparing appropriate probes and primers based on the information, and screening a cDNA library in which the gene is predicted to exist using them.

[0017] In addition, the method for isolating a protein that expresses antimalarial activity according to the present invention is derived from a malaria parasite parasitized in erythrocytes and has antimalarial activity by affinity with a compound represented by the formula (1). A method for isolating expressed protein, wherein a mouth gelinine skeleton is used as a ligand site, a affiliate gel covalently linked to an agarose gel is prepared, and a malaria parasite-derived protein mixture is adsorbed to a powerful affinity gel, A method that fractionates fractions using affinity differences between protein gel and each protein, and that fractions are separated by SDS-polyacrylamide gel electrophoresis to isolate proteins that express antimalarial activity. Affinity gels that are not restricted include, for example, the method of Kawakami et al. (Kawakami, M .; Suzuki, N .; Sudo, Y .; Shishido, T .; Maeda, M. Analytica C himica Acta 1998, 362, 177-186).

[0018] In the formula (1) of the present invention, R \ R ² and R ³ have a hydrogen atom, an unsubstituted or substituted group! Alternatively, it may have a C1-C8 alkyl group, unsubstituted or substituted, or may have a C2-C6 alkyl group, unsubstituted or substituted, or C6. Represents a C10 aryl group, an unsubstituted or substituted heterocyclic group, and R \ R ² and R ³ may be the same or different. The C1-C8 alkyl group may be a straight chain or a branched chain, and may be a methyl group, an ethyl group, an n propyl group, an isopropyl group, an n butyl group, an s butyl group, t Butyl group, isobutyl group, n pentyl group, s-pentyl group, isopentyl group, neopentyl group, n-hexyl group, 2-methyl-n pentyl group, n-heptyl group, 2-methyl-n-hexyl group, n —Octyl group and the like can be exemplified, and examples of the C2-C6 alkenyl group include a buyl group, an aryl group, an isopropenyl group, a butenyl group, which may have a branched chain as well as a straight chain. And C 6 -C 10 aryl groups include phenyl and naphthyl groups, and the heterocyclic groups include pyrrolidine and pyrrole. , Pyrazole, imi Dazole, thiazolidine, thiazole, isothiazole, 2-thiazoline, isoxazole, oxazole, 1,2,5 oxaziazole, piperidine, pyridine, piperazine, pyridazine, pyrimidine, pyrazine, 2H-1, 4 thiazine, 4H-1,4 thiazine, monoreforin, 4H— 1,4 oxazine, indoline, isoindoline, indole, isoindole, indazole, 2H—indazole, benzimidazole, benzothiazonole, benzothiazoline, quinoline, isoquinoline, cinnoline, quinazoline , Quinoxaline, phthalazine, 1,8 naphthyridine and the like obtained by removing one hydrogen from the heterocyclic ring.

[0019] Examples of the substituent in the C1-C8 alkyl group and the C2-C6 alkyl group are the same or different and have 1 to 5 substituents such as an alkoxy group, an alkylcarboxoxy group, and an alkylcarboxyl group. -Group, alkoxycarbo group, substituted or unsubstituted rubamoyl group, substituted or unsubstituted amino group, aryl group, aryloxy group, arooxy group, aroyl group, aryloxycarbonyl group, heterocyclic group , Heterocyclic group-substituted oxy group, heterocyclic group-substituted carbonyloxy group, heterocyclic group-substituted carbonyl group, heterocyclic group substitution Represents a substituted oxycarbonyl group, a halogen atom, a hydroxyl group, an oxo group, a thio group, a carboxyl group, a sulfonic acid group, etc., where the alkyl group in the alkoxy group, alkylcarboxoxy group, alkylcarbonyl group, alkoxycarbonyl group Is the same as the definition of the alkyl group, and the aryl group in the aryloxy group, aroyloxy group, aroyl group, and aryloxycarbonyl group is the same as the definition of the allyl, and is a heterocyclic group-substituted oxy group, a heterocyclic group-substituted carbonyl group. The heterocyclic group in the oxy group, heterocyclic group-substituted carbonyl group, and heterocyclic group-substituted oxycarbonyl group has the same definition as the heterocyclic group described above. In addition, the substituents in the substituent ruberamoyl group are the same or different and represent 1 to 2 substituents, and the substituent represents an alkyl group, alkenyl group, aryl group or heterocyclic group as defined above. The substituents in the substituted amino group are the same or different and represent 1 to 2 substituents, and the substituent represents an alkyl group, an alkyl group, an aryl group or a heterocyclic group as defined above. The halogen atom represents each atom of fluorine, chlorine, bromine and iodine.

[0020] Further, the substituents in the aryl group and heterocyclic group of C6 to C10 are the same or different and have 1 to 5 substituents, for example, an alkyl group, an alkenyl Examples of groups include alkyl groups and alkenyl groups as defined above for alkyl groups and alkenyl groups.

[0021] In the formula (1) of the present invention, A is a nitrogen-containing heterocycle in which a nitrogen atom is bonded to the α-position with respect to the carbon atom bonded to the rhodanine ring, and the nitrogen-containing heterocycle represented by Β It is preferably a 5-membered or 6-membered nitrogen-containing heterocyclic ring which may be the same as or different from the ring. Further, sulfur, oxygen, selenium, etc. are located in the _α position with respect to the carbon atom bonded to the dandanine ring. Of these, nitrogen-containing heterocycles to which oxygen group atoms are bonded are preferable, and nitrogen-containing heterocycles to which sulfur atoms and oxygen atoms are bonded are particularly preferable. The nitrogen-containing heterocyclic ring represented by における in the powerful formula (1) includes pyrrolidine, pyrrole, pyrazole, imidazole, thiazolidine, thiazole, isothiazole, 2-thiazoline, isoxazole, and oxazole, as described above. 1, 2, 5 oxadiazole, etc., piperidine, pyridine, piperazine, pyridazine, pyrimidine, pyrazine, 2H-1, 4 thiazine, 4H-1, 4-thiazine, morpholine, 4H-1, 4— Oxazine and the like can be exemplified. Examples of the substituent bonded to the carbon atom constituting the 5-membered or 6-membered nitrogen-containing heterocycle include the heterocyclic ring. A substituent having the same meaning as the substituent of the group can be exemplified, and the powerful substituent of the nitrogen-containing heterocyclic ring may form an ortho-fused ring with the nitrogen-containing heterocyclic ring. Specifically, the nitrogen-containing heterocycle represented by A in the formula (1) in the case where the substituent formed forms an ortho-fused ring with the nitrogen-containing heterocycle, specifically includes indoline, isoindoline, indole, isoform. Indole, indazole, 2H indazonole, benzoimidazole, benzothiazonole, benzothiazoline, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, phthalazine, 1,8 naphthyridine, etc. can be indicated in the f column.

[0022] In the formula (1) of the present invention, the nitrogen-containing heterocyclic ring represented by B may be the same as or different from the nitrogen-containing heterocyclic ring represented by A. It is preferably a containing heterocyclic ring. n is preferably a conjugated system representing an integer of 0, 1 or 2. Specific examples of the nitrogen-containing heterocycle represented by B in the powerful formula (1) include the same nitrogen-containing heterocycle as the nitrogen-containing heterocycle represented by A in the formula (1) above. I can do it. Specific examples of the substituent bonded to the carbon atom constituting the 5-membered or 6-membered nitrogen-containing heterocycle include the same substituents as those in the nitrogen-containing heterocycle represented by A described above. The powerful nitrogen-containing heterocycle substituents may form ortho-fused rings with nitrogen-containing heterocycles. Specific examples of the nitrogen-containing heterocycle represented by B in the formula (1) in the case where the substituent that forms an ortho-fused ring with the nitrogen-containing heterocycle include the above-mentioned A in the formula (1). The nitrogen-containing heterocyclic ring represented by these can be illustrated.

[0023] Further, among the compounds represented by the formula (1) of the present invention, pharmaceutically acceptable ions are shown, and halogen ions, sulfonate ions, sulfamate ions, hydroxide ions, etc. Specifically, examples of halogen ions include chlorine ions, bromine ions, iodine ions, etc. Examples of sulfonate ions include methanesulfonate ions, ethanesulfonate ions, Examples thereof include aliphatic and aromatic sulfonate ions such as trifluoromethanesulfonate ion, p-toluenesulfonate ion, naphthalenesulfonate ion, 2-hydroxyethanesulfonate ion, and the like. Can be exemplified by cyclohexanesulfamic acid ion, methyl sulfate ion and ethyl sulfate ion. Sulfate ion etc., there may be mentioned hydrogen sulfate ion, borate ion, Al kill and dialkyl phosphate ions, carboxylate ions, carbonate ions, etc. wear. Preferred specific examples of pharmaceutically acceptable ions include chloride ion, bromide, iodine ion, acetate ion, propionate ion, valerate ion, citrate ion, maleate ion, fumarate ion, and lactic acid. Ion, succinate ion, tartrate ion, benzoic acid, methane sulphonic acid, ethanes sulphonic acid, p torenos sulphonate, hydroxide ions, etc. p Toluenesulfonic acid ion, chlorine ion, or hydroxide ion is preferred.

[0024] Specific examples of the compound represented by the above formula (1) include the following. That is, 2-[{5- (3-Methyl 2 (3H) -benzothiazolylidene) 4-oxo-3- (2-Probe) -2 thiazolididene} methyl] -1-methylpyridi -Um = p-toluenesulfonate (MKH-57), 2 [one (3-methyl-2 (3H) -benzothiazolidylidene) 4 oxo3 ethyl 2 thiazolididylidene} methyl] 1 ethylpyridyum Chloride (MKT-077), 4- [One (3-Methyl 2 (3H) benzothiazolidelidene) -4-Exo-3 ethyl) -2 Thiazolididylidene} methyl] 1-Methylquinolium = 1 toluenesulfonate Can be mentioned.

[0025] The method for producing the oral dacyanin dye compound represented by the general formula (1) is not particularly limited, and can be produced by a known method. For example, a derivative in which a leaving group such as a methylthio group is introduced into the nitrogen-containing heterocyclic compound represented by A in General Formula (1), for example, as shown in Formula (2) , 2- (methylthio) benzothiazole (a) was converted to N-methyli derivative (b) using methyl p-toluenesulfonate in a sol-solvent with p-methyl methyl toluenesulfonate. In the presence, it is converted to a merocyanine compound (d) in which an oral danine ring is introduced by reacting with an oral danine ring compound, for example, 3-ethylrodanine (c). Further, the merocyanine compound (d) is a conjugated compound represented by B in the general formula (I) after thiomethylation using p-toluenesulfonic acid methyl ester to convert it to the derivative ( _e ). Nitrogen-containing heterocycles such as N-methylpicolinium p-toluenesulfonate (f), etc. g) can be obtained with a total yield of 38%.

[0026] [Chemical 4]

(g) x = Cl IRA- 00

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

Example 1

[0028] Affi-Tigel represented by the formulas (3) to (6) is the method of Kawakami et al. (Kawakami, M .; Suzu ki, N .; Sudo, Υ ·; Shishido, Τ ·; Maeda, Μ. Analytica Chimica Acta 1998, 362, 177-186) and synthesized. In addition, it was confirmed by UV absorption (490 nm) derived from the dye that oral dashianin was supported on the agarose gel. The affinity gel represented by the formula (3) is a known compound. The affinity gels represented by the formulas (4) to (6) are unknown, including the synthesis method.

[0029] [Chemical 5]

[0030] [6]

[0033] A method for synthesizing the affinity gel represented by formula (4) is shown in formula (7). (A-1) synthesized with reference to the method described in the literature (Satz inger, G. Liebigs Ann. Chem. 1978, 473-511) was reacted with methyl iodide to produce N-methyli A-2) was obtained. Subsequently, by adding the compound (A-3) and triethylamine to (A-2), the oral dasyanin dye (A-4) was obtained in a yield of 90%. The resulting oral dasyanin dye (A-4) is hydrolyzed! /, (A-5), and then condensed with ω-aminobutyragarose gel to give the affine dye represented by formula (4). -Converted to Tiger (Α—6).

[0034] [Chemical 9]

A-1 A-2

A-5

The affinity gel represented by formula (5) and formula (6) was prepared in the same manner as the synthesis method of the affinity gel of formula (4).

[0036] The affinity gel represented by the formulas (3) to (6) obtained in Example 1 was subjected to an isolation experiment for a protozoan protein having a high affinity for oral dasyanin.

Example 2

[0037] Affinity chromatography was examined using the affinity gel represented by the formula (3) prepared in Example 1. In other words, it was decided to search for a target protein by extracting a protein having a strong affinity for the oral dasyanin pigment, which is a ligand. As the malaria parasite, Plasmodium berghei was used.

[0038] The following reagents were used.

PBS (-): 137 mM NaCl (Nacalai Tesque), 2.7 mM KCl (Nacalai Tesque), 8.1 mM Na HPO 12 H O (Nacalai Tesque), 1.5 mM KH PO (Wako Chemicals)

2 4 2 2 4 Saponin supplemented PBS (―): PBS (―), 0.15% saponine (Nacalai Tesque) protease inhibito r: DTT (Nacalai Tesque), PMSF (Nacaiai Tesque), aprotinin (Sigma), leupeptin (Si gma), pepstatin A (Sigma) final concentrations are ImM, 0.5 mM, 2 μg / mL, 2 μg / mL, 2 μg / mL, respectively. [0039] According to the operation procedure shown in Fig. 1, crude malaria parasite protein was separated.

1) From 0.6 to 1.2 mL of blood was collected by heart blood sampling from mice infected with malaria (ICR, o ⁷¹ , 5 weeks, Infected with P. berghei (40% to 70%)).

2) Centrifugation was performed at 800xg for 10 minutes to remove serum, and red blood cells were taken out.

3) Thereafter, saponin-added PBS (-) was added to the blood to dissolve the red blood cells, and the supernatant was removed by centrifugation at 600 xg for 10 minutes. After repeating the same operation three times, washing was carried out by adding the same amount of PBS (-) as before and centrifuging at 600 xg for 10 minutes, and the malaria parasite was taken out from the red blood cells.

4) PBS (-), which was 1/3 of the blood sampled, was added to the protozoa that had been washed three times, added with a protease inhibitor, and ultrasonically ground (10 amp, 5 min, 0 ° C). The supernatant protein (crude protein) was collected by centrifugation at 13000 × g for 10 minutes.

5) The obtained supernatant protein was quantified. The crude protein was used as it was in the affinity-take-matography experiment. Protein quantification was performed by a Bradford method using Protein assay kit (Bio-Rad) and BS A as a standard protein.

[0040] SDS-polyacrylamide electrophoresis was performed using the following reagents.

lx Running Buffer: 25mM Tns (Nacaiai Tesque), 250mM glycine (Nacaiai Tesque), 0.1% SDS (Nacaiai Tesque)

[0041] 0. 1% SDS-containing 12.5% SDS-polyacrylamide gel (acrylamide (Nacaiai Tesque): bis-acrylamide (Nacaiai Tesque) = 30: 0.4) or 15% SDS-polyacrylamide gel ( 29: 1) was prepared and left at 1 ° C to 4 ° C. The next day, each sample was loaded into the gel sample well and run in the lx Running Buffer at 60V for the first time, and then at 160V after entering the separation gel.

[0042] The following reagents were used in the experiment of the affinity-take mouth matography.

Affi-Tigel: Affi-Tigel represented by formula (3)

ω-Aminobutyragarose (Sigma)

Malaria parasite-derived protein: Malaria parasite crude protein isolated in (0039)

Erythrocyte-derived protein: (0039) Protein extract obtained by using non-infected mice instead of malaria-infected mice sample buffer (x5): 0. 3M Tris / HCl (Nacalai Tesque) pH 6. 8,40% glycerol (Nacal ai Tesque), 3.8% DTT, 10% SDS

Washing column: 1000 μL Diamond Precision Tip (Gilson) with moderately packed Quartz Wool (Tosoh)

elution buffer: PBS (—), 50mM MKT— 077

[0043] [Chemical 10]

M KT-077

[0044] According to the operation procedure as shown in Fig. 2, a malaria parasite protein having an affinity for the compound represented by the formula (1) was isolated.

1) Add 100 L of malaria parasite-derived protein with protein concentration adjusted to 5. Omg / mL to the affee-gel (100 L) shown in formula (3) and shake at 30 ° C for 30 minutes . (As a control experiment, ω-aminobutylagarose was used as the affinity gel, and mouse erythrocyte-derived protein was used instead of protozoan-derived protein).

2) Immediately after that, the column was assembled and washed with PBS (-) to remove free protein.

3) After washing, the affinity gel was collected in a 1.5 mL tube, and 100 mL of elution buffer was added. After shaking at 4 ° C for 30 minutes, centrifuge at 10000 X g for 10 minutes, and collect the supernatant protein. Collected.

4) 25 µL of sample buffer (x5) was added to 100 µL of the obtained sample, boiled for 5 minutes, and an appropriate amount of supernatant was collected.

5) They were electrophoresed on SDS-PAGE (12.5%) (60V 45min and 160V 60min). After completion, the gel was cut out and stained with silver (Silver Stainll Kit Wako: Wako Chemicals).

[0045] Fig. 3 shows the results of SDS-PAGE after affee-take mouth matography.

Using a affatigel represented by formula (3) carrying a mouth dashianine dye, the adsorbed crude protein derived from malaria parasite was separated by SDS-P AGE, and 10-20 several bands were observed. (Line 1). In the experiment using malaria-uninfected erythrocytes (line 2) and the experiment using gel without rhodacyanin (line 3), the protein obtained from linel was derived from protozoan malaria. It is concluded that the protein has affinity for oral dasyanin. An experiment to narrow down the protein having higher affinity for oral dasyanin from these several dozen proteins is described in the following example.

Example 3

[0046] The same chromatographies as in Example (2) were performed using the affinity gels represented by formulas (4) to (6). As a comparative control, affi-gel represented by the formula (3) was used.

[0047] Fig. 4 shows the results of SDS-PAGE after affee-take mouth matography. First, we tried to recover the proteins adsorbed on each of the four affinity gels with 0.5 mM eluate, and no protozoan protein was eluted with any affinity gel force (linel, 4, 7, 10). . Next, when elution was performed with 5 mM eluate, the same protein was eluted at almost the same rate for each affinity gel force (lines 2, 5, 8, 11). Finally, when the protein remaining in the affinity gel was collected with a 50 mM concentrated eluate, the same protein was eluted at almost the same rate (lines 3, 6, 9, 12). That is, it became clear that any affinity gel represented by the formulas (3) to (6) can be used to obtain a protein having a high affinity for oral dasyanin. However, as a result of comparing the density of the separated protein bands, some proteins (for example, about 27 kilodaltons) have a higher concentration when using the affinity gel represented by formula (3). It can be seen that the amount can be isolated. Example 4

[0048] By studying the method of elution of the protein adsorbed on the affinity gel, it was decided to narrow down the high affinity protein. In the process of elution, ideally, forces with a large dissociation constant can be eluted in sequence with the affair gel. As a result, the target protein was isolated by selecting the protein with the smallest dissociation constant. That is, when the adsorbed protein was eluted, a gradient column method was applied in which a gradient was applied to the concentration of the eluate.

[0049] The following materials were used as experimental materials.

Affi-Tigel: Affi-Tigel represented by formula (3)

elution buffer A: PBS (—), lmM MKT— 077

elution buffer B: PBS (—), 50 mM MKT— 077

[0050] The experimental procedure was as follows.

1) To 100 L of Affi-Tigel, 100 L of protein from malaria parasite with a protein concentration of 5. Omg / mL was added and shaken at 4 ° C for 30 minutes.

2) After adsorption, the column was assembled and washed with PBS (-) to remove free protein.

3) After washing, pour 80 / zL of low concentration eluate (elution buffer A) into the column and pour the eluate from the column into a 1.5mL tube 33 times. Subsequently, elution was performed 6 times with 80 μL of high-concentration eluate (elution buffer B).

4) After adding sample buffer (x5) to each obtained fraction, it was boiled for 5 minutes and an appropriate amount of supernatant was collected.

5) Finally, electrophoresis was performed with SDS-PAGE (12.5% or 15%), and after completion, the gel was cut out and stained with silver.

[0051] FIG. 5 shows the results of SDS-PAGE of each fraction eluted with the gradient column. Some proteins were recovered by high concentration eluate (fractions 34-40). Among the collected proteins, five proteins of 27, 29, 31, 33, and 37 kDa were obtained with high reproducibility and high specificity. In particular, the 33,31,27 kDa proteins were always obtained in this experiment and were found to be particularly specific for the oral dasyanin dye.

Example 5 [0052] (Isolation of protein for analysis)

A protein band having the highest affinity with the oral dasyanin dye obtained in Example 4 (0051)

Then, it was cut out from the SDS-PAGE separation gel and subjected to sequence analysis.

[0053] According to Example 4, SDS-PAGE was performed. In the silver staining method, 0.2 gZmL sodium thiosulfate (Wako Chemicals) was used instead of the sensitizing solution.

[0054] The target protein band (37, 33, 31, 29, 27 kDa) stained by silver staining was cut out and sampled in Eppendorf tubes washed three times with acetonitrile.

[0055] Among the five isolated proteins, a partial amino acid sequence was determined for the 27,33 kDa protein. The method was CID-MS / MS. As a result, it became clear that CNNNNNNNEK had a 27 kDa protein and TIDNNNIDEK had a 33 kDa protein, and a 33 kDa protein. Each of these proteins is rich in arginine, demonstrating that it is a malaria protein.

Industrial applicability

[0056] According to the present invention, it is possible to develop a drug having a high affinity for a mouth dassanini compound and targeting a protein or a gene encoding the same.

Claims

Claims An isolated protein derived from Plasmodium sp. Parasitized on erythrocytes and expressing antimalarial activity by affinity with a compound represented by formula (1).

[Chemical 1]

(In the formula, R \ R ² and R ³ each represent a hydrogen atom, an unsubstituted or substituted alkyl group, an alkyl group, an aryl group, or a heterocyclic group, which may be the same or different. A and B represent a nitrogen-containing heterocyclic ring, n represents an integer of 0, 1 or 2, and represents a pharmaceutically acceptable ion.

[2] The isolated protein according to claim 1, which has a partial amino acid sequence of TIDNNNIDEK

[3] The isolated protein according to claim 2, wherein the size determined by SDS-polyacrylamide gel electrophoresis is approximately 33 kilodaltons.

[4] A gene encoding the isolated protein according to claim 2 or 3.

5. The isolated protein according to claim 1, which has a partial amino acid sequence of CNNNNNNNEK.

6. The isolated protein according to claim 5, wherein the size determined by SDS-polyacrylamide gel electrophoresis is approximately 27 kilodaltons.

[7] A gene encoding the isolated protein according to claim 5 or 6.

[8] SDS—size determined by polyacrylamide gel electrophoresis is approximately 29 kiloda 2. The isolated protein of claim 1 which is a ruton.

The isolated protein of claim 1, wherein the size determined by SDS-polyacrylamide gel electrophoresis is approximately 31 kilodaltons.

The isolated protein of claim 1, wherein the size determined by SDS-polyacrylamide gel electrophoresis is approximately 37 kilodaltons.

A method for isolating a protein derived from Plasmodium sp. Parasitic on erythrocytes and expressing antimalarial activity by affinity with a compound represented by formula (1), wherein the oral dasocyanin skeleton is a ligand site The affiliation gel is covalently bonded to the affiliation gel, the protein mixture derived from the malaria parasite is adsorbed to the powerful affinity gel, and the difference in affinity between the affinity gel and each protein is used for fractionation. A method for isolating a protein that expresses antimalarial activity, comprising separating the protein that expresses antimalarial activity by SDS-polyacrylamide gel electrophoresis.

12. The method for isolating a protein that expresses antimalarial activity according to claim 11, wherein the protein has a partial amino acid sequence of TIDNNNIDEK that expresses antimalarial activity.

13. The method for isolating a protein that expresses antimalarial activity according to claim 12, wherein the size determined by SDS-polyacrylamide gel electrophoresis is approximately 33 kilodaltons.

12. The method for isolating a protein that expresses antimalarial activity according to claim 11, wherein the protein has a partial amino acid sequence of CNNNNNNNEK that expresses antimalarial activity.

The method for isolating a protein that expresses antimalarial activity according to claim 14, wherein the size determined by SDS-polyacrylamide gel electrophoresis is about 27 kilodaltons.

12. The method for isolating a protein that expresses antimalarial activity according to claim 11, wherein the size determined by SDS-polyacrylamide gel electrophoresis is about 29 kilodaltons.

The protein power for expressing antimalarial activity The size determined by SDS-polyacrylamide gel electrophoresis is about 31 kilodaltons. Of a protein that expresses

[18] The method for isolating a protein expressing antimalarial activity according to [11], wherein the size determined by SDS-polyacrylamide gel electrophoresis is approximately 37 kilodaltons.

[19] Claims 1-3, 5, 6, 8-: The isolated protein according to any one of LO is brought into contact with the test substance, and the affinity between the isolated protein and the test substance is determined. A method for screening a substance having antimalarial activity, characterized by measuring and evaluating the degree.