JP2005087012A - Method for testing inhibitory ability for proliferation of plasmodium species - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method, etc., for testing the inhibitory ability for proliferation of Plasmodium species which is essential to searching for substances having the inhibitory ability for the proliferation of the Plasmodium species. <P>SOLUTION: The method for testing the inhibitory ability for the proliferation of the Plasmodium species comprises the following steps. (1) a first step of assaying the activity of a triglyceride synthase selected from a protein, etc., composed of a specific amino acid sequence derived from the Plasmodium species in a contact system of the triglyceride synthase with a test substance and (2) a second step of evaluating the inhibitory ability of the substance for the proliferation of the Plasmodium species based on the difference obtained by comparing the activity assayed in the first step with the activity in the control. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for assaying malaria parasite growth inhibitory ability and the like.

Malaria, which is characterized by regular fever, severe anemia, and spleen enlargement, includes the genus Plasmodium (eg, Plasmodium falciparum, P. vavax, P. malariae, etc.), a protozoan protozoan. It is a disease caused by protozoa as a parasitic pathogen, and is still widespread worldwide, mainly in the tropical region. The onset of malaria is caused by the repetition of a proliferation cycle in red blood cells after a malaria parasite that has entered the human body via an anopheles passes through a latent period in the liver. Drugs such as chloroquine are the most commonly used drugs for treating malaria, but their effectiveness has been losing in recent years due to the emergence of drug-resistant and multi-drug resistant strains, and there is no effective antimalarial vaccine is the current situation.
Recent studies have shown that malaria parasites are rapidly synthesizing triglycerides at specific times of growth, so triglycerides are considered to be an essential factor for the growth and survival of malaria parasites. The final stage of triglyceride synthesis is catalyzed by diacylglycerol acyltransferase (hereinafter referred to as DGATase). The membrane fraction of Plasmodium has DGATase activity, and it has been reported that a drug that inhibits rat DGATase significantly suppresses the growth of Plasmodium in malaria-infected erythrocytes (see Patent Document 1).
On the other hand, since DGATase of plasmodium and the gene encoding DGATase were unknown, it was difficult to easily screen a drug directly acting on DGATase enzyme of malaria from a wide range of test substances.
JP 2003-189897 A

  An object of the present invention is to provide a simple method for testing malaria parasite growth inhibitory ability, which is essential for searching for substances having the ability to inhibit malaria parasite growth.

As a result of intensive studies under such circumstances, the present inventors have found that the malaria parasite gene having the base sequence represented by SEQ ID NO: 1 is a malaria parasite DGATase. That is, the protein (SEQ ID NO: 2) encoded by the gene having the base sequence represented by SEQ ID NO: 1 had triglyceride synthase (DGATase) activity. The present invention has been completed based on the above findings.
That is, the present invention
[1] An assay method for malaria parasite growth inhibitory ability,
(1) a first step of measuring the activity of the triglyceride synthase in a contact system of a triglyceride synthase selected from the following protein group and a test substance, and (2) an activity and a control measured in the first step A second step of evaluating the ability of the substance to inhibit malaria parasite growth based on the difference obtained by comparing the activity in
A method for assaying the ability to inhibit the growth of Plasmodium, characterized by comprising:
<Protein group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2, comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted, and triglyceride synthesis ability (C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having the ability to synthesize triglyceride (d) a DNA having a base sequence represented by SEQ ID NO: 1 A protein comprising the amino acid sequence encoded by (e) consisting of an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 1, and comprising triglyceride synthesis A protein having the ability (f) having the base sequence represented by SEQ ID NO: 1 Evaluation by the assay method described in the protein [2] [1], which comprises a DNA having a complementarity to the DNA and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions and has the ability to synthesize triglycerides A method of searching for a malaria parasite growth inhibitory substance, comprising selecting a substance having a malaria parasite growth inhibitory capacity based on the malaria parasite growth inhibitory capacity,
[3] A malaria parasite growth inhibitor comprising, as an active ingredient, a substance selected by the search method according to [2] or a pharmaceutically acceptable salt thereof;
[4] A protein having any of the following amino acid sequences;
<Amino acid sequence group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2, comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted, and triglyceride synthesis ability (C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having the ability to synthesize triglyceride (d) a DNA having a base sequence represented by SEQ ID NO: 1 A protein comprising the amino acid sequence encoded by (e) consisting of an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 1, and comprising triglyceride synthesis A protein having the ability (f) having the base sequence represented by SEQ ID NO: 1 The amino acid sequence of the protein according to [5] [4], which comprises a DNA having a complementarity to a DNA and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions and has the ability to synthesize triglycerides A gene encoding
[6] A catalyst for the following reaction comprising the protein according to [4];
1,2-diacylglycerol + acyl-CoA ⇒ triglyceride + CoA
[7] Use of the protein according to [4] as a reagent that provides an index for evaluating the ability to inhibit malaria parasite growth;
[8] A gene containing the base sequence represented by SEQ ID NO: 1;
[9] A gene containing the base sequence represented by SEQ ID NO: 3;
Etc. are provided.

  According to the present invention, it has become possible to provide a simple method for testing malaria parasite growth inhibitory ability, which is essential for searching for substances having the ability to inhibit malaria parasite growth.

The present invention is described in detail below.
Triglyceride synthase (DGATase) is an enzyme called alias 1,2-diacylglycerol acyltransferase, and catalyzes the following reaction (hereinafter sometimes referred to as the present enzyme).

1,2-diacylglycerol + acyl CoA ト リ triacylglycerol + CoA

The activity of such an enzyme can be measured, for example, by the following method (for example, the method described in JP-A-8-18296, Shinsei Kagaku Kenkyu 4 Lipid I, page 85, etc.).
Specifically, for example, after radiolabeling one of 1,2-diacylglycerol and acyl CoA, which are substrates for the above reaction, triglyceride synthase (DGATase) is added to the reaction solution containing both substrates. Added. After a certain time, the produced radiolabeled triglyceride is quantified by usual radioactivity measurement.
As 1,2-diacylglycerol, 1,2-diacylglycerol containing 2 or more saturated or unsaturated fatty acids having 3 or more carbon atoms as an acyl group is used, and radiolabeled 1,2-diacylglycerol is used. In the 1,2-diacylglycerol, one containing one or more tritium or ¹⁴ C in the glycerol part or the fatty acid part may be used.
As the acyl CoA, a saturated or unsaturated fatty acid having 3 or more carbon atoms and CoA (coenzyme A) that is thioester-bonded is used. A material containing the above tritium or ¹⁴ C may be used.

Examples of the reaction solution include 0 mM to 10 mM (preferably 1 mM to 5 mM) ethylenediaminetetraacetic acid, 0 mM to 200 mM (preferably 20 mM to 100 mM) magnesium ion, 0 μM to 100 μM (preferably 1.8 μM to 18 μM) bovine serum. A buffer solution (preferably 10 mM to 200 mM Tris-HCl buffer) having a pH of about 6 to 8 (preferably pH 7.5 to 8.0) to which albumin, 0 M to 1 M sucrose, or the like is added may be used.
The reaction temperature can be 10 ° C. to 40 ° C. (preferably 37 ° C.).
The reaction time is about 1 minute or more (preferably 5 minutes to 1 hour).
Extraction and quantification of the radiolabeled triglyceride produced in the reaction solution may be performed using, for example, an ordinary method as described in Examples described later.

  In the present specification, the enzyme of the present invention represents a malaria parasite-derived triglyceride synthase selected from the following (a) to (e). That is, (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence represented by SEQ ID NO: 2, comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted, and triglyceride A protein having synthetic activity, (c) a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having triglyceride synthetic activity, (d) a base shown in SEQ ID NO: 1 A protein comprising an amino acid sequence encoded by a DNA having a sequence; (e) an amino acid sequence encoded by a DNA having a base sequence having 80% or more sequence identity with the DNA having the base sequence represented by SEQ ID NO: 1 And a protein having triglyceride synthesis activity, (f) represented by SEQ ID NO: 1 A DNA having complementarity to DNA having the nucleotide sequence consists of the amino acid sequence encoded by a DNA hybridizing under stringent conditions, and may be mentioned a protein having a triglyceride synthesis activity.

Here, “deletion, addition or substitution of amino acids” in (b) and “80% or more sequence identity” in (c) and (e) are represented by SEQ ID NO: 2, for example. This includes a process that a protein having an amino acid sequence undergoes in a cell, a naturally occurring mutation due to a species difference, an individual difference, a tissue difference, or the like of an organism from which the protein is derived, or an artificial amino acid mutation.
As a technique for artificially performing the “deletion, addition or substitution of amino acids” in the above (b) (hereinafter sometimes collectively referred to as amino acid modification), for example, the amino acid represented by SEQ ID NO: 2 is used. There is a technique in which conventional site-directed mutagenesis is performed on DNA encoding the sequence, and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer Etc.
The number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be in a range in which DGATase activity can be found.
Of the deletions, additions or substitutions, the modification relating to amino acid substitution is particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and structural features. Such substitutions include, for example, i) glycine, alanine; ii) valine, isoleucine, leucine; iii) aspartic acid, glutamic acid, asparagine, glutamine, iv) serine, threonine; v) lysine, arginine; vi) phenylalanine, Substitution within the tyrosine group.
In the present invention, “sequence identity” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENETYX-MAC, or a public database, for example, the homepage address http It is generally available at: //www.ddbj.nig.ac.jp.
The sequence identity in the present invention may be 80% or more, preferably 90% or more, more preferably 95% or more.
Regarding “hybridize under stringent conditions” in (f) above, the hybridization used here is, for example, by Sambrook J., Frisch EF, Maniatis T., Molecular Cloning Second Edition (Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory issuance (Cold Spring Harbor Laboratory press), etc. “Stringent conditions” means, for example, 6 × SSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10 × SSC), 45% in a solution containing 50% formamide. Examples include conditions (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6) that form a hybrid at 50 ° C and then wash with 2 × SSC at 50 ° C. it can. The salt concentration in the washing step can be selected, for example, from 2 × SSC at 50 ° C. (low stringency conditions) to 0.2 × SSC at 50 ° C. (high stringency conditions). The temperature in the washing step can be selected, for example, from a temperature from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.

A method for preparing the enzyme of the present invention will be described below.
First, a gene comprising a base sequence encoding the amino acid sequence of the enzyme of the present invention (hereinafter sometimes referred to as the present gene), for example, (I) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, II) A gene having the nucleotide sequence shown in SEQ ID NO: 1 is converted into a conventional genetic engineering method (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Spring. Acquired according to the method published by the Harbor Laboratory (Cold Spring Harbor Laboratory press). Alternatively, it is possible to artificially synthesize a target base sequence according to a generally used oligonucleotide synthesis method. Next, by using the obtained gene of the present invention, the present enzyme is produced and obtained according to a normal genetic engineering method. In this way, the enzyme of the present invention can be prepared.

  For example, it can be obtained by preparing a plasmid that allows the gene of the present invention to be expressed in a host cell, introducing it into the host cell, transforming it, and then culturing the transformed host cell (transformant). What is necessary is just to acquire this invention enzyme from a culture.

This transformed cell can be prepared as follows.
A plasmid is prepared by inserting the gene of the present invention so as to be connected to a promoter in a form that can be expressed in a vector that can be used in a cell into which the gene of the present invention is introduced, using ordinary genetic engineering techniques. The promoter used here may be any one that can function in the cell into which the present gene is introduced. When the cell is an animal cell, the SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Examples include Virus promoter (RSV promoter), β-actin gene promoter, aP2 gene promoter and the like. A commercially available vector containing such a promoter upstream of the multicloning site may be used.
Examples of the plasmid include a promoter that contains genetic information that can be replicated in a host cell, can be propagated autonomously, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those in which a gene comprising a base sequence encoding the amino acid sequence of the enzyme of the present invention is introduced into an expression vector having a detectable marker. Various types of expression vectors are commercially available. For example, an expression vector used for expression in E. coli is an expression vector containing a promoter such as lac, trp, tac, etc., and these are commercially available from Pharmacia, Takara Shuzo and others. Restriction enzymes used for introducing a gene comprising a base sequence encoding the amino acid sequence of this enzyme into the expression vector are also commercially available from Takara Shuzo.
When it is necessary to induce higher expression, a ribosome binding region may be linked upstream of a gene consisting of a base sequence encoding the amino acid sequence of this enzyme. Examples of the ribosome binding region used include those described in reports by Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).

The plasmid is then introduced into the cell. Examples of the introduction method into the cell include a calcium phosphate method, an electric introduction method, a DEAE dextran method, and a micelle formation method. As the calcium phosphate method, the method described in Grimm, S. et al., Proc. Natl. Acad. Sci. USA, 93, 10923-10927, etc., as the electrophoretic method and the DEAE dextran method, Ting, AT et al., The method described in EMBO J., 15, 6189-6196 etc., and the micelle formation method is the method described in Hawkins, CJ et al., Proc. Natl. Acad. Sci. USA, 93, 13786-13790 etc. Can be mentioned. When using the micelle formation method, commercially available reagents such as Lipofectamine (manufactured by Gibco) and Fugene (manufactured by Boehringer) may be used.
The transformed cells are selected by culturing the cells subjected to the plasmid introduction treatment, for example, using a selection marker gene previously contained in the vector and culturing in a medium under selection conditions corresponding to the selection marker gene. be able to. Further, selection may be continued to obtain the present transformed cell that has become a stable transformant in which this gene is introduced into the chromosome. In order to confirm that the introduced gene has been incorporated into a chromosome present in the cell, the genomic DNA of the cell is prepared according to a normal genetic engineering method and has a partial base sequence of the gene. The presence of the present gene in the genomic DNA may be detected and confirmed using a method such as PCR using DNA as a primer or Southern hybridization using a DNA having a partial base sequence of the present gene as a probe.

Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, or mammalian cells. For example, Escherichia coli and the like can be preferably mentioned from the viewpoint that mass preparation of the present enzyme becomes easy.
The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culturing is performed in a medium appropriately containing an appropriate carbon source, nitrogen source, and micronutrients such as vitamins. The culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration and agitation culture.
The acquisition of this enzyme may be carried out in combination with methods commonly used for isolation and purification of general proteins. For example, the transformants obtained by the above culture are collected by centrifugation or the like, and the transformants are crushed or dissolved, and if necessary, proteins are solubilized, and various types such as ion exchange, hydrophobicity, gel filtration, etc. What is necessary is just to refine | purify the process using a chromatography individually or in combination. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and the enzyme may be purified from the culture supernatant in the same manner as described above. If necessary, an operation for restoring the higher-order structure of the purified protein may be further performed.

The assay method of the present invention comprises (1) a step of measuring the triglyceride synthase (DGATase) activity in a cell-free system in a contact system between triglyceride synthase (DGATase) and a test substance, and (2) measurement by the above step. Evaluating the ability of the substance to inhibit the growth of Plasmodium based on the difference obtained by comparing the activity obtained and the activity in the control.
In the assay method of the present invention, the rate of inhibition of triglyceride synthase (DGATase), which serves as an index for evaluating the ability to inhibit the growth of malaria parasites, is observed without directly observing the proliferation of malaria parasites in cells (details will be described later). The ability of the test substance to inhibit malaria parasite growth can be evaluated. For this reason, in this method, screening can be easily performed based on a specific action while targeting a wide range of test substances, and is optimal for primary screening and the like.

The form of the enzyme that is contacted with the test substance in the first step of the assay method of the present invention may be (A) a purified product, a crude product, or the like of the enzyme, or (B) contained in the cell. When the test substance is contacted with a transformed cell into which the enzyme (for example, a gene comprising a base sequence encoding the amino acid sequence of the enzyme has been introduced (hereinafter sometimes referred to as the transformed cell)) Or the like. Furthermore, the cells may be cells separated from the tissue, or may be cells in a state forming a population having the same function / morphology.
For example, in the case of (A) above, the concentration of the present enzyme to be contacted with the test substance is, for example, 5 ng / ml or more, preferably 1 μg / ml or more.
In the case of (B) above, the concentration of the enzyme to be contacted with the test substance is, for example, 1 × 10 ⁵ cells / ml or more, preferably 1 × 10 ⁶ cells / ml or more as the concentration of the transformed cells. It is.

In this step, for example, (A) a form in which three kinds of substrates (for example, 1,2-diacylglycerol and fatty acid CoA) and a test substance are in contact with the enzyme, and (B) two kinds of substrates One of them (for example, 1,2-diacylglycerol or fatty acid CoA) and the test substance are in contact with this enzyme (ie, the test substance is handled as the other of the two substrates) ), The activity of the enzyme may be measured in any form.

Furthermore, when measuring the activity of the present enzyme in the above-mentioned form, the order in which the test substance and the substrate are brought into contact with the enzyme is (a) first contacting the enzyme with the test substance and keeping the temperature constant for a certain period of time. A form in which two or one kind of substrate is added, (b) a form in which the present enzyme, test substance and two or one kind of substrate are contacted simultaneously, (c) two or one kind of the present enzyme and Any form of adding the test substance after first contacting the substrate and keeping the temperature constant for a certain period of time may be used.
In the case of (a), the contact time between the present enzyme and the test substance can be, for example, 1 minute or longer (preferably 1 minute or longer and within 1 hour). As a heat retention temperature in the said contact system, 10 to 50 degreeC (preferably 20 to 40 degreeC) can be mention | raise | lifted, for example.
In the case of the above (c), examples of the heat retention temperature in the contact system include 10 ° C. to 50 ° C. (preferably 20 ° C. to 40 ° C.).

  When measuring the activity of the enzyme in the above form, the concentration of the test substance in the contact system between triglyceride synthase (DGATase) and the test substance is, for example, when the test substance is a low molecular weight compound, 0.01 μM to 50 mM (preferably 0.1 μM to 50 mM) can be mentioned. Examples of the substrate concentration include 1 μM to 10 mM (preferably 10 μM to 1 mM). On the other hand, the concentration of the enzyme to be contacted with the test substance can be, for example, 10 ng / ml or more (preferably 1 μg / ml or more).

When measuring the activity of the enzyme in the above-mentioned form, for example, after reacting a mixed solution containing the enzyme, the test substance and the substrate for a certain period of time, the amount of reduction in the raw materials such as the substrate in the reaction is analyzed. Or a method for analyzing an increase in the amount of product in the reaction. These methods may be any known activity measurement method. Specific examples include the methods described in JP-A-8-18296, Shinsei Kagaku Kogaku Kenkyu 4 Fat I, page 85, etc. as described above.
When measuring the activity of the present enzyme in the above form, the reaction temperature at the time of measuring the activity can be, for example, 10 ° C. to 50 ° C. (preferably 20 ° C. to 40 ° C.). Examples of the reaction time include 1 minute or more (preferably 5 minutes to 1 hour). As reaction pH, 6.5-9 (preferably pH 7.5-8) can be mention | raise | lifted, for example.

  For detection of a raw material such as a substrate or a product in an enzyme reaction or measurement of the amount thereof, the reaction solution is subjected to, for example, high performance liquid chromatography (hereinafter referred to as HPLC), thin layer chromatography, paper chromatography or the like. After separation, UV absorption or radioactivity of the raw material or product is detected (when the raw material previously labeled with a radioisotope is used), or UV absorption or radioactivity is measured. Such a method may be used. When it is necessary to remove the protein from the reaction solution at the time of the above analysis, the protein may be removed from the reaction solution by a method described in, for example, Methods Enzymol. 280, 211-221.

Based on the difference obtained by comparing the activity measured as described above with the activity in the control (ie, reference substance, negative control, etc.), the ability of the substance to inhibit malaria parasite growth is evaluated. In this way, the test for the ability of the test substance to inhibit the growth of Plasmodium can be performed (the assay method of the present invention).
In the assay method of the present invention, when the measured activity and the activity in the control are compared, at least one of the two or more different substances is a substance (for example, a solvent) that has no ability to inhibit malaria parasite growth. , Negative control such as a test system solution as a background), the ability of the other test substance to suppress the growth of malaria parasite may be evaluated, and at least of the two or more different substances described above The malaria parasite growth inhibitory ability of the other test substance may be evaluated based on the malaria parasite growth inhibitory ability of one substance (for example, a reference substance). Of course, both may be evaluated.

In the case of measuring the activity of the enzyme in the above-described form, for example, the first step of the assay method of the present invention is to comprise two kinds of substrates (for example, 1,2-diacylglycerol and fatty acid CoA) and a test substance. In the case where the three are in contact with this enzyme (ie, (A) above), and when both negative control and reference substance (eg, standard substrate) are used as controls in the process Using the activity measurement value of triglyceride synthase (DGATase), the inhibition rate may be determined according to the following formula.

Inhibition rate (%) = {control (negative control) value−measurement (test substance) value} × 100 / control (negative control) value

And what is necessary is just to evaluate the malaria parasite growth suppression capability with the calculated inhibition rate. On the other hand, the first step of the assay method of the present invention is a mode in which three substrates, ie, two kinds of substrates (for example, 1,2-diacylglycerol and fatty acid CoA) and a test substance are in contact with the enzyme (ie, the above (A) ) And when using a substance (reference substance) that has the ability to inhibit the growth of Plasmodium as a control in the process, compare the control (reference substance) value with the measured (test substance) value. What is necessary is just to evaluate the malaria parasite growth inhibitory ability. In this case, if the measured (test substance) value is lower than the control (reference substance) value, the test substance is evaluated as having a higher ability to suppress the growth of Plasmodium than the reference substance.
In addition, for example, the first step of the assay method of the present invention is a form in which one of two substrates (for example, 1,2-diacylglycerol or fatty acid CoA) and a test substance are in contact with the enzyme ( That is, in the case of (B) above, in the case where the test substance is handled as the other of the two kinds of substrates), a substance having a malaria parasite growth inhibitory ability as a control (reference substance) And the control (reference substance) value and the measured (test substance) value may be compared to evaluate the ability to inhibit malaria parasite growth. In this case, if the measured (test substance) value is higher than the control (reference substance) value, the test substance is superior to the reference substance as a substrate for the enzyme. It will be superior to the reference substance as a competitive inhibitor of this enzyme.

In order to search for a malaria parasite growth inhibitory substance (that is, an antimalarial therapeutic substance), a substance having a malaria parasite growth inhibitory ability may be selected based on the malaria parasite growth inhibitory ability evaluated by the assay method of the present invention (this book Invention Search Method).
For example, in the case of (A) above and when a negative control is used as a control, the inhibition rate serving as an index for evaluating the ability of the test substance to inhibit the growth of malaria parasite is a statistically significant value. More specifically, for example, a substance having an inhibition rate of 30% or more in the above formula, more preferably a substance showing 50% or more, is selected as a substance having the ability to inhibit malaria parasite growth. On the other hand, in the case of (A) above, when a substance (reference substance) having malaria parasite growth inhibitory ability is used as a control, the measured (test substance) value is lower than the control (reference substance) value. The substance which shows is selected as a substance which has the malaria parasite growth inhibitory ability. Further, for example, in the case of (B) above, a substance having a measured (test substance) value higher than a control (reference substance) value is selected as a substance having ability to inhibit malaria parasite growth.
The above-mentioned “substance with the ability to suppress malaria parasite growth” is any substance such as a low molecular weight compound, a polynucleotide, a protein or a peptide, as long as it has the ability to reduce the triglyceride synthase activity of the enzyme of the present invention. Also good.
More specifically, for example, (a) a low molecular compound that reduces the triglyceride synthase activity of the enzyme of the present invention, (b) an antisense nucleic acid of the gene of the present invention, (c) an antibody that specifically binds to the enzyme of the present invention. Etc.

  The substance selected by the search method of the present invention has a malaria parasite growth inhibitory activity and can be used as an active ingredient of an antimalarial therapeutic agent.

  As described above, the triglyceride synthase of the present invention can be used as a reagent that provides an index for evaluating the ability to inhibit the growth of malaria parasites.

A malaria parasite growth inhibitor comprising a substance having an ability to inhibit triglyceride synthase (DGATase) or a pharmaceutically acceptable salt thereof as an active ingredient (that is, an antimalarial therapeutic agent: hereinafter, the inhibitor of the present invention and The effective amount can be orally or parenterally administered to mammals such as humans. For example, when administered orally, the inhibitor of the present invention can be used in a usual form such as a tablet, capsule, syrup, suspension or the like. In addition, when administered parenterally, the inhibitor of the present invention can be used in the form of a usual solution such as a solution, emulsion, suspension or the like. Examples of a method for parenteral administration of the inhibitor of the present invention include a method of injection, a method of administering to the rectum in the form of a suppository, and the like.
The appropriate dosage form is a substance having a substance capable of inhibiting triglyceride synthase or a pharmaceutically acceptable salt thereof in an acceptable normal carrier, excipient, binder, stabilizer, diluent and the like. It can manufacture by mix | blending. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.
The dose varies depending on the age, sex, weight, disease level, type of inhibitor of the present invention, dosage form, etc. of the mammal to be administered. About 1 mg to about 2 g, preferably about 5 mg to about 1 g as the amount of active ingredient, and in the case of injection, about 0.1 mg to about 500 mg as an active ingredient may be administered as an adult. In addition, the above-mentioned daily dose can be administered once or divided into several times.

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

(Extraction of sequences represented by SEQ ID NOs: 1 and 2 (protozoan DGAT1))
Using the full length part of the mouse DGAT1 amino acid sequence (GenBank ID number: AAC72917.1) and the human DGAT1 amino acid sequence (GenBank ID number: AAC63997.1), an amino acid sequence highly homologous to this amino acid sequence is NCBI (http: / Extracted from the amino acid sequence database of /www.ncbi.nlm.nih.gov/entrez/query.fcgi) by the tblastn program. As a result, the amino acid sequence shown in SEQ ID NO: 2 was extracted as a candidate amino acid sequence of Plasmodium DGAT1, and the gene sequence encoding this amino acid sequence was further extracted as a candidate gene sequence (SEQ ID NO: 1) of Plasmodium DGAT1 from the NCBI gene database. Extracted.

(Acquisition of oligonucleotide consisting of the base sequence of SEQ ID NO: 3)
In order to increase the expression efficiency of the gene represented by SEQ ID NO: 1 in mammalian cells, the base sequence represented by SEQ ID NO: 3 was designed. Specifically, the base sequence of SEQ ID NO: 3 was designed by replacing the codon in SEQ ID NO: 1 with another codon encoding the same amino acid (eg, AAT → AAC). An oligonucleotide consisting of a sequence obtained by adding the ACGCGTGCCCACC sequence (SEQ ID NO: 4) to the 5 ′ end and the TAAGCGGCCCGC sequence (SEQ ID NO: 5) to the 3 ′ end of the base sequence shown in SEQ ID NO: 3 Acid Synthesizer, Applied Biosystems). From the nucleotide sequence of SEQ ID NO: 3 by Sanger's method (F. Sanger, S. Nicklen, AR Coulson, Proceedings of National Academy of Science USA (1977), 74, 5463-5467). The nucleotide sequence of the oligonucleotide was determined. An E. coli plasmid prepared by subcloning the synthesized oligonucleotide into pT7-Blue vector (Novagen). E. coli JM109 strain competent cell (Toyobo) was transformed. By isolating and purifying the plasmid from the cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN), SEQ ID NO: 3 A plasmid containing an oligonucleotide having a base sequence was obtained.

(Preparation of a plasmid expressing a protein having the amino acid sequence shown in SEQ ID NO: 2)
The plasmid obtained in Example 2 was cleaved with restriction enzymes MluI and NotI, and inserted into the similarly cleaved pAP3neo plasmid (Takara Shuzo). A plasmid prepared by subcloning with E. coli. E. coli JM109 strain competent cell (Toyobo) was transformed. By isolating and purifying the plasmid from the cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN), SEQ ID NO: 2 A plasmid was obtained that expressed a protein having the amino acid sequence shown (hereinafter also referred to as PfDGAT1 protein).

(Preparation of cells expressing PfDGAT1 protein)
The PfDGAT1 protein expression plasmid obtained in Example 3 was introduced into CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) using a LipofectAMINE PLUS reagent kit (Invitrogen) according to the procedure attached to the reagent kit. The CHO-K1 transformed cells into which the expression plasmid was introduced were cultured for 24 hours according to the protocol distributed at the time of purchase of the cells.

(Preparation of negative control cells)
As a control (negative control), a transformed cell (control) was prepared in the same manner as described in Example 4 except that pAP3neo (not containing the PfDGAT insertion sequence) was used instead of the PfDGAT1 protein expression plasmid. Obtained.

(Preparation of enucleated cell extract containing this enzyme)
After culturing the transformed cells obtained in Examples 4 and 5, the cells were washed twice with D-PBS (Gibco), then scraped with a cell scraper, and centrifuged to collect the cells. The collected cells were treated with 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1 mM ethylene glycol bis 2 aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), 1/100 volume protease inhibitor cocktail ( The sample was suspended in a buffer solution (hereinafter referred to as Buffer A) containing Sigma. The suspension was subjected to ultrasonic treatment (under ice cooling, 5 seconds × 6 times) to disrupt the cells in the suspension, and then the resulting cell disruption solution was treated at 1000 × g for 30 seconds at 4 ° C. Centrifugation removed unbroken cells and nuclear fractions as precipitates. About the obtained supernatant, what measured the density | concentration of the protein by the Bradford method which used bovine serum albumin as a standard was used as the enucleated cell extract.

(Measurement of diacylglycerol acyltransferase activity)
10 μL of the enucleated cell extract obtained in Example 6 was added to 150 μL of a reaction buffer solution (0.25 M sucrose, 100 mM Tris-hydrochloric acid (pH 7.5), 20 mM magnesium chloride, 1 mM EDTA, 1.25 mg / mL. After suspending in bovine serum albumin, 0.5 mM 1,2-dioleoylglycerol, 0.67% DMSO, 30 [mu] M (0.03 [mu] Ci) [< ¹⁴ > C] palmitoyl-coenzyme A), the mixture is allowed to stand at room temperature for 10 minutes. Keep warm. In addition, the test system which performed operation similar to the above using the buffer solution for reaction which is the same said composition except not containing 1, 2- dioleoylglycerol was made into the background section.
Next, 300 μL of heptane / 2-propanol / water (= 80: 20: 2) was added to the mixture and stirred, allowed to stand for 10 minutes, and then 200 μL of heptane and 100 μL of water were further added to the mixture and stirred. did. After stirring, the mixture was centrifuged to remove the lower layer, and the remaining upper layer was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in chloroform. Next, this solution is spotted on a glass plate for silica gel 150 thin layer chromatography (K5 silica gel 150 Å, Whatman) and developed using a developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1). Thus, [ ¹⁴ C] triacylglycerol contained in the solution was separated. The separated [ ¹⁴ C] triacylglycerol was quantified using a bioimaging analyzer system BAS-2000 (Fuji Film).
FIG. 1 shows the activity of diacylglycerol acyltransferase in each enucleated cell extract in a test system in which a control (negative control) plasmid was introduced and a test system in which a plasmid for expressing PfDGAT1 protein was introduced.

As described above, the activity of the diacylglycerol acyltransferase in the enucleated cell extract was increased in the test system in which the plasmid for expressing the PfDGAT1 protein was introduced, compared to the test system in which the plasmid for the control (negative control) was introduced. From this, it was confirmed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 exhibits the activity of diacylglycerol acyltransferase.

(Measurement of triglyceride synthesis in cells)
The cells obtained in Examples 4 and 5 were cultured in a 6-well plate until confluent. The medium of this cell was F-12 medium (Sigma) containing 10% bovine serum, 10 ng / mL bovine insulin, final concentration of 6.45 μM [U- ^14C ] D-glucose (Amersham, radioactive 4 mCi / L). After the replacement, the cells were cultured at 37 ° C. for 4 hours. The cultured cells were washed once with PBS buffer, and the cells were treated with 1 ml of heptane / isopropanol (2: 1) for 10 minutes. The heptane / isopropanol solution containing triglyceride in the cells was collected with a pipette, and the collected solution was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in 30 μl of chloroform / methanol (2: 1, v / v). This solution was then spotted onto a silica gel 150 thin layer chromatography glass plate (K5 silica gel 150 Å, Whatman). A developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1, v / v) was put in a sealed container, and the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature. The dried TLC plate was exposed to an imaging plate (Fuji Film Co.) for 16 hours. After completion of the exposure, the imaging plate was analyzed with a bioimage analyzer (BAS 2500, Fuji Film), and the [ ¹⁴ C] radioactivity of the portion corresponding to the development position of the triacylglycerol on the TLC plate was measured.
FIG. 2 shows the measurement results of the amount of triglyceride synthesized in each cell in the test system into which the control (negative control) plasmid was introduced and the test system into which the plasmid for expressing PfDGAT1 protein was introduced.

As a result, the amount of triacylglycerol synthesized in the cells was significantly increased in the test system in which the plasmid for expressing the PfDGAT1 protein was introduced, compared to the test system in which the control (negative control) plasmid was introduced. From these results, it was confirmed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 contributes to triglyceride synthesis in the cell.

(Example of screening for Plasmodium DGAT1 (PfDGAT1) inhibitor)
Inhibitors of the enzyme of the present invention (PfDGAT1) are disclosed in, for example, J. Org. B. Screening can be performed according to the method of Rodgers et al. (Journal of Lipid Research, Vol. 10, p427-432). In this method, the SH group of coenzyme A released by a reaction catalyzed by DGAT is reacted with 5,5′-dithiobis (2-nitrobenzoic acid) (hereinafter referred to as DTNB) to quantify the color development of the reaction product. It is a method to do. Specifically, 10 μL of the enucleated cell extract of PfDGAT1-expressing cells obtained in Example 6 was added to 300 μL of a reaction buffer (67 mM) containing a test substance (1% dimethyl sulfoxide (hereinafter referred to as DMSO) solution). After suspension in Tris-hydrochloric acid (pH 8.0), 1 mM DTNB, 0.67% bovine serum albumin, 0.1 mM 1,2-dioleoylglycerol, 0.67% DMSO, 30 μM palmitoyl-coenzyme A) The mixture is kept warm at 30 ° C. In the test substance-free group, the same method as described above was used except that only 1% DMSO was added instead of the DMSO solution of the test substance. In addition, in the background group, the same method as the test substance and 1,2-dioleoylglycerol was added except that only DMSO was added instead of the test substance and 1,2-dioleoylglycerol. Using.
Ten minutes after the start of the reaction, the reaction is stopped by adding a 100 mM sodium acetate solution to the reaction solution, and the absorbance at 412 nm is measured. The measured value in the test substance addition group is defined as the activity of diacylglycerol acyltransferase (referred to as measured value A) when the test substance is added. For the test substance-free group and the background group, the DGATase activity is measured using the same method, and the measurement value B and the measurement value C are obtained, respectively. From these measured values, it is possible to calculate the test substance's ability to inhibit diacylglycerol acyltransferase as an inhibition rate ((measured value B−measured value A) × 100 / (measured value B−measured value C)).

  According to the present invention, it has become possible to provide a simple method for testing malaria parasite growth inhibitory ability, which is essential for searching for substances having the ability to inhibit malaria parasite growth.

The activity of diacylglycerol acyltransferase was measured on enucleated cell extracts of cells (Vec) into which a control (negative control) plasmid was introduced and cells into which a plasmid for expressing PfDGAT1 protein (PfDGAT1) was introduced. It is a figure which shows the DGAT activity at the time of a substrate no addition (DMSO) and a substrate addition (1,2-DOG) of DGAT, respectively. It is a figure which shows the result of having measured the triglyceride synthesis amount by a glucose label | marker about the cell (Vec) by which the plasmid of control (negative control) was introduce | transduced, and the cell (PfDGAT1) by which the plasmid for PfDGAT1 protein expression was introduce | transduced.

SEQ ID NO: 3
Variant of the gene described in SEQ ID NO: 1 SEQ ID NO: 4
Restriction enzyme: MluI recognition sequence and Kozak sequence SEQ ID NO: 5
Stop codon and restriction enzyme: NotI recognition sequence

Claims (7)

A test method for malaria parasite growth inhibition ability,
(1) a first step of measuring the activity of the triglyceride synthase in a contact system of a triglyceride synthase selected from the following protein group and a test substance, and (2) an activity and a control measured in the first step A second step of evaluating the ability of the substance to inhibit malaria parasite growth based on the difference obtained by comparing the activity in
A method for assaying the ability to inhibit the growth of Plasmodium parasites, comprising:
<Protein group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2, comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted, and triglyceride synthesis ability (C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having the ability to synthesize triglyceride (d) a DNA having a base sequence represented by SEQ ID NO: 1 A protein comprising the amino acid sequence encoded by (e) consisting of an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 1, and comprising triglyceride synthesis A protein having the ability (f) having the base sequence represented by SEQ ID NO: 1 That a DNA having complementarity to DNA, consisting of an amino acid sequence encoded by a DNA hybridizing under stringent conditions, and protein having a triglyceride synthesis ability A method for searching for a malaria parasite growth inhibitory substance, comprising selecting a substance having a malaria parasite growth inhibitory ability based on the malaria parasite growth inhibitory ability evaluated by the assay method according to claim 1. A malaria parasite growth inhibitor comprising, as an active ingredient, a substance selected by the searching method according to claim 2 or a pharmaceutically acceptable salt thereof. A protein having any one of the following amino acid sequences:
<Amino acid sequence group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2, comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted, and triglyceride synthesis ability (C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having the ability to synthesize triglyceride (d) a DNA having a base sequence represented by SEQ ID NO: 1 A protein comprising the amino acid sequence encoded by (e) consisting of an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 1, and comprising triglyceride synthesis A protein having the ability (f) having the base sequence represented by SEQ ID NO: 1 That a DNA having complementarity to DNA, consisting of an amino acid sequence encoded by a DNA hybridizing under stringent conditions, and protein having a triglyceride synthesis ability The gene which codes the amino acid sequence of the protein of Claim 4. A catalyst for the following reaction, comprising the protein according to claim 4.
1,2-diacylglycerol + acyl-CoA ⇒ triglyceride + CoA Use of the protein according to claim 4 as a reagent providing an index for evaluating the ability to inhibit the growth of Plasmodium.

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