JP4722512B2 - Screening method for inhibitors of γ-glutamylcysteine synthetase - Google Patents

Description

  The present invention relates to a screening method for inhibitors of γ-glutamylcysteine synthetase. In addition, this application is an application of the result which concerns on national commission.

  Homeostasis by glutathione plays an important role in maintaining intracellular redox balance and maintaining defense against oxidative stress and chemical stress. On the other hand, control of glutathione levels has attracted pharmacological attention. This is because glutathione is involved in the mechanism of acquiring multidrug resistance in certain parasites and cancer cells. When an anticancer agent or an antiparasitic agent invades cancer cells or parasite cells, these agents and glutathione are combined to form a GSH inclusion compound, and this GSH inclusion compound is, for example, an MDR transporter. It is discharged out of the cell by etc. By such a mechanism, parasites and cancer cells acquire drug resistance.

γ-Glutamylcysteine synthetase (glutamate-cysteine ligase: EC 6.3.2.2, hereinafter referred to as γGCS) is an enzyme that catalyzes the first step of glutathione biosynthesis. The glutathione biosynthesis system is shown in FIG. As shown in FIG. 3, GanmaGCS is, Mg ^2+, in the presence ATP, from L- glutamic acid and L- cysteine, catalyzes a reaction γ- Guru Tamil cysteine is synthesized. This reaction catalyzed by γGCS is the rate-limiting step of glutathione biosynthesis. In addition, the activity of γGCS is precisely regulated in vivo by non-allosteric feedback inhibition by glutathione, lack of intracellular L-cysteine, and transcriptional and post-transcriptional expression control under various physiological conditions. Yes.

  ΓGCS is attracting attention as a drug discovery target for reasons such as glutathione's involvement in the mechanism of acquiring multidrug resistance. An example of a traditional γGCS inhibitor is L-butionine sulfoximine (BSO). BSO can prolong the life of mice infected with Trypanosoma brucei, a parasite parasite that causes human sleep sickness (see Non-Patent Document 1), and can inhibit the growth of Plasmodium falciparum. (See Non-Patent Document 2). Furthermore, it has been reported that pretreatment of cancer cells with the BSO can remove drug resistance due to MRP transporters in the cancer cells (see, for example, Non-Patent Documents 3 and 4).

Other γGCS inhibitors include compounds having a sulfoxyimine structure as in the case of BSO, and consisting of an analog of a predicted transition state of γ-glutamyl phosphate and L-cysteine (non-patent) Reference 5).
BA Arrick, OW Griffith, A. Cerami, J. Exp. Med. 153, 720 (1981). K. Luersen, RD Walter, S. Muller, Biochem. J. 346, 545 (2000). G. Rappa et al., Eur. J. Cancer 39, 120 (2003). T. Fojo, S. Bates, Oncogene 22, 7512 (2003). J. Hiratake, T. Irie, N. Tokutake, J. Oda, Biosci. Biotechnol. Biochem. 66, 1500 (1998).

  However, as an inhibitor of γGCS, for example, there is no known inhibitor that specifically acts on γGCS of non-human organisms including the parasites, and a method capable of screening for such inhibitors is desired. ing.

  Accordingly, an object of the present invention is to provide a screening method for inhibitors of γGCS specific to non-human organisms including parasites.

In order to achieve the above object, the screening method of the present invention is a method for screening an inhibitor of γ-glutamylcysteine synthetase (γGCS), which is a human type γGCS shown in (A) below and a non-screening agent shown in (B) below. Preparing a human-type γGCS, bringing a drug candidate compound prepared in advance into contact with each of the human-type and non-human-type γGCS, and evaluating the strength of binding of the compound to both γGCS, The method includes a step of selecting the drug candidate compound that binds more strongly to the non-human γGCS than to the type γGCS.
(A) Human-type γGCS: an amino acid residue located at the L-cysteine binding site of γGCS, the amino acid residue corresponding to position 132 of the amino acid sequence when aligned with the amino acid sequence of SEQ ID NO: 1, ΓGCS, which is arginine.
(B) Non-human γGCS: an amino acid residue located at the L-cysteine binding site of γGCS, and when aligned with the amino acid sequence of SEQ ID NO: 1, the amino acid residue corresponding to position 132 of the amino acid sequence is ΓGCS, which is an amino acid residue other than arginine.

The present inventors are compounds that bind to γGCS and have a sulfoxyimine structure that is an analog of a transition state between γ-glutamyl phosphate, which is a reaction intermediate of γGCS, and L-cysteine. Focusing on the fact that the portion corresponding to the carboxyl group and the side chain affects the strength of the bond between the compound and γGCS, intensive research was repeated. The present inventors first, E. coli GanmaGCS: A (AC No.P06980 SEQ ID NO: 1) and co-crystals of the compound represented by the following structural formula (1), the crystalline space group is P2 ₁ The crystal structure of the γGCS was clarified for the first time in the world. Next, the present inventors have found that although one of the amino acid residues contributing to the L-cysteine binding site of γGCS revealed by the structural analysis is conserved in many γGCSs including humans, Focusing on the substitution in certain types of parasites, it has been found that based on the structural differences caused by the substitution of the amino acid residues, compounds can be designed that are specific inhibitors of the parasite γGCS. It was. Furthermore, the present inventors have found that the designed drug candidate compound can be screened by using the recombinant γGCS substituted with the amino acid residue, and have reached the present invention.

  According to the screening method of the present invention, for example, inhibitors specific to non-human γGCS including γGCS of parasites such as Trypanosoma brucei and Plasmodium falciparum can be screened. Since the inhibitor can suppress the acquisition of drug resistance by the parasite, it can be used, for example, for a new therapeutic / prophylactic drug for a parasitic infection or a therapeutic / preventive method.

  In one aspect, the screening method of the present invention uses a γGCS inhibitor of Trypanosoma brucei as a screening target, and as the non-human γGCS, the amino acid residue corresponding to the 132nd position is asparagine. This is a method using human γGCS.

  The screening method of the present invention uses, as another aspect, a non-human γGCS in which an inhibitor of γGCS of Plasmodium falciparum is a screening target and the amino acid residue corresponding to the 132nd amino acid is glutamine. It is a method to do.

  In the screening method of the present invention, as the drug candidate compound, a compound designed from crystal structure analysis data of a co-crystal of E. coli γGCS and the compound represented by the structural formula (1) can be used. Among these, as the drug candidate compound, it is preferable to use a compound having a chemical structure represented by the following general formula (2).

In the above formula (2), X represents —H, —CH ₃ , —CH ₂ CH ₃ , —CH ₂ OH, —CH ₂ SH, —CH ₂ NH ₂ , —CH ₂ NHCH ₃ , —CH ₂ N ( _{CH 3) 2, -CF 3,} -CH 2 Br, -CH 2 Cl, -CHCl 2, -CN, -NC, -CH 2 CN, -CH 2 NC, -CH 2 CH 2 CH 3, -CH 2 _{CH 2 OH, -CH 2 CH 2} SH, -CH 2 CH 2 Cl, -CH 2 CH 2 Br, -CH 2 CF 3, -COOH, -CH 2 COOH, -COCH 3, -COCH 3, -PO 3 H, —CH ₂ PO ₃ H, —P (CH ₃ ) O ₂ H, —SO ₃ H, —SO ₂ H, —NO ₂ , —C≡CH, —CH═CH ₂ , —CONH ₂ , —CH ₂ COCNH _2, and a substituent selected from the group consisting of -CH ₂ CH ₂ NH _2, Y is, -H, -OH, -COOH, _{NH 2, -COOCH 3, -CONH 2} , -CN, -SO 3 H and, a substituent selected from the group consisting of -NO _2.

  In the screening method of the present invention, the strength of binding between γGCS and the drug candidate compound can be evaluated by, for example, an assay method based on slow-binding kinetics.

  The non-human recombinant γGCS of the present invention is an amino acid residue located at the L-cysteine binding site of γGCS and corresponds to the 132nd amino acid sequence when aligned with the amino acid sequence of SEQ ID NO: 1. Recombinant γGCS where the residue is an amino acid residue other than arginine. The non-human recombinant γGCS of the present invention can be used, for example, in the screening method of the present invention.

The non-human recombinant γGCS of the present invention is preferably the non-human recombinant γGCS described in (i) or (ii) below.
(I) an amino acid sequence in which the 132nd arginine of E. coli γGCS amino acid sequence (AC No. P06980: SEQ ID NO: 1) is substituted with asparagine or glutamine;
An amino acid sequence in which the 131st arginine of Vibrio cholerae γGCS amino acid sequence (AC No. AAF93724: SEQ ID NO: 2) is substituted with asparagine or glutamine;
Pseudomonas aeruginosa γGCS amino acid sequence (AC No.NP 253890: an amino acid sequence in which the 134th arginine of SEQ ID NO: 3) is substituted with asparagine or glutamine,
Salmonella γGCS amino acid sequence (AC No.NP 457216: an amino acid sequence in which the 132nd arginine of SEQ ID NO: 4) is substituted with asparagine or glutamine,
Bufnella γGCS amino acid sequence (AC No.NP 777980: an amino acid sequence in which the 132nd arginine of SEQ ID NO: 5) is substituted with asparagine or glutamine,
Human γGCS amino acid sequence (AC No.NP 001489: amino acid sequence in which the 191st arginine of SEQ ID NO: 6) is substituted with asparagine or glutamine,
An amino acid sequence in which the 191st arginine of the rat γGCS amino acid sequence (AC No. P97494: SEQ ID NO: 7) is substituted with asparagine or glutamine;
An amino acid sequence in which the 191st arginine of the mouse γGCS amino acid sequence (AC No. O09172: SEQ ID NO: 8) is substituted with asparagine or glutamine;
Mycoplasma γGCS amino acid sequence (AC No.NP 857368: The amino acid sequence in which the 112th arginine of SEQ ID NO: 9) is substituted with asparagine or glutamine, and the 185th arginine of the trypanosoma glue γGCS amino acid sequence (AC No. AAC80009: SEQ ID NO: 10) is asparagine. Or a non-human recombinant γGCS comprising an amino acid sequence selected from the group consisting of amino acid sequences substituted with glutamine.
(Ii) In the amino acid residue other than the amino acid residue substituted with asparagine or glutamine in the amino acid sequence of (i) above, one or several amino acid residues consist of an amino acid sequence deleted, substituted or added. And a non-human recombinant γGCS having γ-glutamylcysteine synthetase activity.

The human recombinant γGCS of the present invention is an amino acid residue located at the L-cysteine binding site of γGCS, and when aligned with the amino acid sequence of SEQ ID NO: 1, the amino acid residue corresponding to position 132 of the amino acid sequence is the amino acid residue. It is a recombinant γGCS whose group is arginine. The human recombinant γGCS of the present invention can be used, for example, in the screening method of the present invention. As the human recombinant γGCS of the present invention, the human recombinant γGCS described in the following (iii) or (iv) is preferable.
(Iii) an amino acid sequence in which the 185th asparagine of trypanosoma blues γGCS (AC No. AAC47195: SEQ ID NO: 11) is substituted with arginine, or
A human-type recombinant γGCS comprising an amino acid sequence in which the 473rd glutamine of P. falciparum γGCS (AC No. T18400: SEQ ID NO: 12) is substituted with arginine.
(Iv) an amino acid residue other than the amino acid residue substituted with arginine in the amino acid sequence of (iii) above, wherein one or several amino acid residues are deleted, substituted or added, and , A non-human recombinant γGCS having γ-glutamylcysteine synthetase activity.

  The polynucleotide of the present invention is a polynucleotide encoding the recombinant γGCS of any one of (i) to (iv) above, and the vector of the present invention is a vector containing the polynucleotide of the present invention. The transformed organism or cell thereof of the present invention is an organism or cell thereof transformed by the vector of the present invention.

The crystal of the present invention is a co-crystal used for designing a drug candidate compound, which is a co-crystal of γGCS comprising the amino acid sequence of SEQ ID NO: 1 and the compound represented by the structural formula (1), The group is P2 ₁ .

  The method for producing a prophylactic / therapeutic agent for parasitic infection according to the present invention comprises a step of preparing a drug candidate compound, and a compound that specifically inhibits non-human γGCS by the screening method of the present invention using the drug candidate compound. And a process for producing a prophylactic / therapeutic agent for parasitic infections containing an effective amount of the selected compound.

  First, γGCS used in the screening method of the present invention will be described. In the present invention, human-type γGCS is an amino acid residue located at the L-cysteine binding site of γGCS and corresponds to the 132nd amino acid residue of the amino acid sequence when aligned with the amino acid sequence of SEQ ID NO: 1. Refers to γGCS, which is arginine. Many organisms including humans, rats, and bacteria have human-type γGCS. Table 1 below shows organisms having human type γGCS, database registration numbers of the respective γGCS sequences, and positions of amino acid residues corresponding to the 132nd arginine.

(Table 1)
Organism Registration number Sequence position Sequence number <br/> Escherichia coli P06980 132 1
Vibrio cholerae AAF93724 131 2
Pseudomonas aeruginosa NP 253890 134 3
Salmonella enterica NP 457216 132 4
Buchnera aphidicola NP 777980 132 5
Human NP 001489 191 6
Rat P97494 191 7
Mouse O09172 191 8
Mycoplasma (Mycobacterium bovis AF2122 / 97) NP 857368 112 9
Trypanosoma cruzi AAC80009 185 10

  In the screening method of the present invention, the non-human γGCS is an amino acid residue located at the L-cysteine binding site of γGCS and corresponds to the 132nd amino acid sequence when aligned with the amino acid sequence of SEQ ID NO: 1. Refers to γGCS, in which the amino acid residue is an amino acid residue other than arginine. The organisms inherently having non-human γGCS include those shown in Table 2 below. Table 2 below shows organism names, sequence database registration numbers, amino acid residues corresponding to the 132nd position, and their positions.

(Table 2)
Organism registration number Corresponding amino acid residue Sequence position SEQ ID NO <br/> Trypanosoma blues
(Trypanosoma brucei) AAC47195 Asparagine 185 11
Plasmodium falciparum
(Plasmodium falciparum) T18400 Glutamine 473 12

  The present inventors conducted three-dimensional structure analysis of Escherichia coli γGCS and revealed for the first time in the world the three-dimensional and two-dimensional structures of γGCS shown in FIGS. 1A and 1B, respectively. Furthermore, the present inventors performed multiple alignment with various γGCSs based on the results of the above three-dimensional structure analysis, and formed an N-terminal variable arm (105th position of SEQ ID NO: 1) that constitutes the L-cysteine binding site in γGCS. ˜145th) and the central variable arm (the 238th to 311th of SEQ ID NO: 1) were successfully aligned. An example of the alignment is shown in FIG. FIG. 2 shows Escherichia coli (Ec), Vibrio cholerae (Vc), Pseudomonas aeruginosa (Pa), Brunella (Ba), human (Hu), rat (Ra), trypanosoma cruzi (Tc), trypanosome blues (Tb) , And alignment for P. falciparum (Pf). As shown in FIG. 2, the 132nd arginine (R) of Escherichia coli (Ec), which was revealed to be an amino acid residue involved in the recognition of the side chain of L-cysteine from the three-dimensional structure analysis, was trypanosome blues ( It is preserved except for Tb) and P. falciparum (Pf). The 132nd arginine (R) is replaced with asparagine (N) in trypanosoma blues, and with glutamine (Q) in P. falciparum.

  Therefore, as the non-human γGCS, the trypanosoma blues γGCS or the non-human group in which arginine (R) corresponding to the 132nd amino acid residue in the human γGCS is substituted with asparagine (N) If the modified γGCS is used, a γGCS inhibitor specific to trypanosome blues can be screened.

  In addition, as non-human γGCS, γGCS of P. falciparum or non-human arginine (R) corresponding to the 132nd amino acid residue in human γGCS is replaced with glutamine (Q). If recombinant γGCS is used, a γGCS inhibitor specific to P. falciparum can be screened.

  The screening target of the screening method of the present invention is not limited to the trypanosoma blues or plasmodium falciparum γGCS inhibitor, and if the γGCS is naturally non-human, it is a γGCS inhibitor specific to that organism. Can be screened. Whether γGCS of a given organism is human or non-human is determined by, for example, referring to FIG. 2 and performing multiple alignment and determining whether the amino acid residue corresponding to position 132 of SEQ ID NO: 1 is arginine it can.

The drug candidate compound used in the screening method of the present invention is not particularly limited, and any chemical substance can be used as a candidate compound. Among them, the drug candidate compound is preferably designed based on the γGCS three-dimensional structure information obtained from the crystal analysis data of the crystal of the present invention. The design of drug candidate compounds based on the three-dimensional structure information can be performed, for example, by searching for a compound that matches the active center of the enzyme from a low molecular compound library by a docking simulation method using the following software. One aspect thereof is a method of constructing a pharmacophore model by combining with, for example, a drug candidate compound as a lead compound of a sulfoximine type inhibitor. Examples of the software include DOCK, AUTODOCK, 3D-Dock, FlexX (above, freeware), trade name: C2.Ludi (Fujitsu, Accelrys Inc.), trade name: AS DOCK (manufactured by Ryoka System Co., Ltd.) and the like. Further, the three-dimensional structure information of γGCS can be obtained by preparing the γGCS crystal of the present invention and analyzing the crystal by X-ray analysis according to the method described in Examples described later.

  The drug candidate compound used in the screening method of the present invention, which is a γGCS inhibitor drug candidate compound specific for Trypanosoma blues or Plasmodium falciparum, is considered to form a recognition site for the L-cysteine side chain. It is preferable to design the molecule so as to include a functional group capable of forming a hydrogen bond with asparagine or glutamine corresponding to the 132nd position of SEQ ID NO: 1.

  Specific examples of drug candidate compounds used in the screening method of the present invention include drug candidate compounds for inhibitors that function as a mechanism-based inhibitor having the chemical structure of the following general formula (2).

In the above formula (2), X represents —H, —CH ₃ , —CH ₂ CH ₃ , —CH ₂ OH, —CH ₂ SH, —CH ₂ NH ₂ , —CH ₂ NHCH ₃ , —CH ₂ N ( _{CH 3) 2, -CF 3,} -CH 2 Br, -CH 2 Cl, -CHCl 2, -CN, -NC, -CH 2 CN, -CH 2 NC, -CH 2 CH 2 CH 3, -CH 2 _{CH 2 OH, -CH 2 CH 2} SH, -CH 2 CH 2 Cl, -CH 2 CH 2 Br, -CH 2 CF 3, -COOH, -CH 2 COOH, -COCH 3, -COCH 3, -PO 3 _{H, -CH 2 PO 3 H,} -P (CH 3) O 2 H, -SO 3 H, -SO 2 H, -NO 2, -C≡CH, -CH = CH 2, -CONH 2, -CH ₂ COCNH _2, and, a substituent selected from the group consisting of _{-CH 2 CH 2 NH 2, -CH} 2 CH 3, -CH 2 OH, _{CH 2 SH, -CH 2 NH 2} , -CH 2 NHCH 3, -CH 2 CN, -CF 3, -CH 2 CF 3, or, preferably -CN, more preferably, -CH ₂ CH _3, or CH ₂ OH. Y is a substituent selected from the group consisting of —H, —OH, —COOH, —NH ₂ , —COOCH ₃ , —CONH ₂ , —CN, —SO ₃ H, and —NO _2. -H, -OH, or -COOH is preferred, and -H or -COOH is more preferred.

  When the compound having the chemical structure of the general formula (2) functions as a mechanism-based inhibitor, after binding to γGCS, it is phosphorylated as in the following general formula (3) and strongly bound to γGCS. It will be converted and will function as an inhibitor.

  The method for bringing the human type γGCS or non-human type γGCS into contact with the drug candidate compound is not particularly limited, and may be an in vitro method or an in vivo method.

  The in vitro method is not particularly limited, and includes, for example, the purified human type or non-human type γGCS, L-glutamic acid, L-cysteine or L-2-aminobutyric acid, ATP, and magnesium. An example is a method in which the drug candidate compound is added to the reaction solution and incubated. In this case, the strength of the binding between the drug candidate compound and γGCS can be measured by the degree of inhibition received by the enzyme activity of γGCS. When a drug candidate compound of an inhibitor that functions as the mechanism-based inhibitor is used, for example, an assay method based on slow-binding kinetics (see, for example, Non-Patent Document 5) etc. The strength of the inhibitory action can be quantified.

Specifically, for example, the reaction with γGCS (E), substrate (glutamic acid: S), product (ADP: P), and inhibitor (I) is represented by the following formula (4), and the product ADP And the rate constant kon [I] or inhibition constant Ki of “EI → EI *” is determined from the following formulas (5) to (7), and the magnitude of the kon or the Ki is determined as the inhibitory action of the inhibitor. There is a method of quantifying the strength. In the following formula, Km represents the Km value for the reaction of “E + S⇔ES” of γGCS.
E + S ＥＳ ES → E + P
↑ ↓ (4)
EI → EI *
[P] = [P] max · [1-exp (−kt)] (5)
k = kon [I] / (1+ [S] / Km) (6)
Ki = [E] · [I] / [EI *] (7)

  The in vivo method is not particularly limited. For example, human and non-human γGCS can be obtained by a method in which a drug candidate compound is allowed to act directly on the parasite having non-human γGCS, a gene recombination technique, or the like. Examples include a method of producing a model experimental organism or cell having only one of them, allowing a drug candidate compound to act on the organism or cell, and bringing it into contact with γGCS inside the cell. As a method for evaluating the strength of binding between the drug candidate compound and γGCS, for example, since glutathione is deficient due to inhibition of γGCS, for example, a reporter is provided downstream of the promoter of a gene induced by glutathione deficiency. An example is a method in which a gene is placed and evaluated using the expression of the reporter gene as an index.

  The human γGCS and non-human γGCS used in the screening method of the present invention preferably have the same origin. This is because it becomes easy to determine the strength of the bond. In that case, the organism to be derived is not particularly limited, and examples thereof include experimental animals that are human γGCS such as Escherichia coli, yeast, insect cells, animal cells, etc., and non-human γGCS corresponding to the human γGCS. The recombinant γGCS of the present invention in which the arginine corresponding to the 132nd position of the human γGCS is substituted. The expression and purification method of these γGCS is not particularly limited, and can be performed by a conventionally known method.

  The non-human recombinant γGCS of the present invention includes a recombinant γGCS in which arginine at the sequence position shown in the same table in the amino acid sequence of γGCS of an organism having a human γGCS shown in Table 1 above is substituted with a different amino acid. . Although it does not restrict | limit especially as a substituted amino acid, Asparagine or glutamine is preferable. The non-human recombinant γGCS of the present invention further comprises an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence other than the substituted amino acid, and γ -Includes non-human recombinant γGCS having glutamylcysteine synthetase activity. In such a non-human recombinant γGCS, the number of amino acid residues to be deleted, substituted or added is not particularly limited as long as γ-glutamylcysteine synthetase activity is not inhibited, but is, for example, 1 to 20 Preferably, it is 1-10, More preferably, it is 1-5, More preferably, it is 1-2. In addition, the non-human recombinant γGCS of the present invention can be used for various protein or peptide tags within a range that does not inhibit enzyme activity for the purpose of simplifying and reducing the purification procedure, improving stability, and solubilizing. And may be fused.

  Specific examples of the non-human recombinant γGCS of the present invention include the non-human recombinant γGCS described in (i) or (ii) above. Further, as shown in FIG. 2, in Trypanosoma blues and Plasmodium falciparum, the 131st amino acid residue of SEQ ID NO: 1 is threonine (T), and thus, for example, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, etc. In non-human recombinant γGCS such as fungus, Salmonella and Buchnera, a non-human recombinant γGCS in which the tyrosine (Y) corresponding to the 131st position of SEQ ID NO: 1 is replaced with threonine (T) is also preferable.

  In the non-human recombinant γGCS of the present invention, for example, the human γGCS coding sequence (CDS) of Table 1 is first cloned, and then arginine at the sequence position of Table 1 is added to the CDS asparagine or A polynucleotide comprising a non-human recombinant γGCS CDS is obtained by introducing a mutation substituting for glutamine, and the CDS is incorporated into an appropriate expression vector and introduced into an appropriate host for expression, and if necessary It can be manufactured by purifying. Those skilled in the art can easily clone the γGCS coding sequence (CDS) of various organisms listed in Table 1 and introduce the substitution mutation. Table 3 below shows the data bank registration numbers of γGCS CDS of various organisms listed in Table 1 and the sequence positions where substitution mutations should be introduced for the production of non-human recombinant γGCS.

(Table 3)
Organism Registration number Sequence position Sequence number E. coli U00096 394-396 13
Vibrio cholerae AE004141 391-393 14
Pseudomonas aeruginosa NC 002516 400-402 15
Salmonella NC 003198 394-396 16
Bufnera NC 004545 394-396 17
Human NM 001498 571-573 18
Rat NM 010295 571-573 19
Mouse NM 008129 571-573 20
Mycoplasma NC 002945 334-336 21
Trypanosome Cluj AF095637 552-555 22

  The polynucleotide of the present invention encoding the non-human recombinant γGCS of the present invention is obtained, for example, as described above, when a polynucleotide comprising human γGCS CDS is obtained and aligned with the amino acid sequence of SEQ ID NO: 1. Can be produced by introducing a mutation into the arginine residue corresponding to position 132 of the amino acid sequence. Conventionally known methods can be applied to the CDS amplification method, cloning method, and site-directed mutagenesis method. As a specific example of the polynucleotide of the present invention, in the base sequence encoding the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 13: E. coli γGCS gene), the 394th to 396th encoding the 132nd arginine residue. Examples include polynucleotides having a base sequence in which the base sequence of “cgt” is replaced with “aac”, “aat”, “caa”, or “cag”.

  The human recombinant γGCS of the present invention is the amino acid of the non-human γGCS in which the amino acid residue corresponding to the 132nd amino acid sequence is an amino acid residue other than arginine when aligned with the amino acid sequence of SEQ ID NO: 1. The residue is an arginine and includes the recombinant γGCS of (iii) and (iv) above. In the human recombinant γGCS (iv), the number of amino acid residues to be deleted, substituted or added is not particularly limited as long as the γ-glutamylcysteine synthetase activity is not inhibited. It is 20, Preferably it is 1-10, More preferably, it is 1-5, More preferably, it is 1-2. In addition, the non-human recombinant γGCS of the present invention can be used for various protein or peptide tags within a range that does not inhibit enzyme activity for the purpose of simplifying and reducing the purification procedure, improving stability, and solubilizing. And may be fused.

  In the human recombinant γGCS of the present invention, for example, the coding sequence (CDS) of the non-human γGCS shown in Table 2 is first cloned, and then asparagine or glutamine at the sequence position shown in Table 2 is added to the CDS. A polynucleotide comprising a human recombinant γGCS CDS is obtained by introducing a mutation that substitutes for arginine, and the CDS is incorporated into an appropriate expression vector and introduced into an appropriate host for expression, and if necessary It can manufacture by refine | purifying. Those skilled in the art can easily clone the γGCS coding sequences (CDS) of various organisms listed in Table 2 and introduce the substitution mutation. Table 4 below shows the data bank registration number of the non-human γGCS CDS in Table 2 and the sequence positions for introducing substitution mutations for the production of human recombinant γGCS.

(Table 4)
Organism Registration number Sequence position SEQ ID NO: Trypanosome Blues U56818 552-555 23
P. falciparum NC 004330 1417-1419 24

  The polynucleotide of the present invention encoding the human recombinant γGCS of the present invention can be produced in the same manner as the above-described method for producing the polynucleotide encoding the non-human γGCS of the present invention.

  The polynucleotide of the present invention can be made into the vector of the present invention by, for example, incorporating it into a conventionally known expression vector or recombinant vector. The vector of the present invention can be used, for example, for producing a non-human recombinant γGCS of the present invention or for producing a transformed organism or its cells for use in an in vivo screening method. Therefore, the vector of the present invention is preferably an expression vector capable of expressing the non-human recombinant γGCS of the present invention in the host to be introduced. The transformed organism or cell thereof of the present invention is an organism or cell thereof transformed by the vector of the present invention. The organism is not particularly limited, and for example, those derived from microorganisms, animals or plants can be used. The production of these organisms or cells thereof is not particularly limited, and can be performed by applying a conventionally known gene transfer technique. The gene introduction method is not particularly limited, and examples thereof include a method using a phage or virus, a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, and a calcium phosphate method. The transformed organism or cell thereof of the present invention can be used, for example, in the screening method of the present invention when evaluating the strength of binding of a drug candidate compound to γGCS in vivo.

The crystal of the present invention is a co-crystal used for designing a drug candidate compound, and is a co-crystal of E. coli γGCS consisting of the amino acid sequence of SEQ ID NO: 1 and the compound represented by the structural formula (1), A crystal whose space group is P2 ₁ .

  The production of the crystal of the present invention has a technical problem that the obtained crystal is not a single crystal but has a twin or polycrystalline structure, and is not suitable for structural analysis. In general, in order to perform structural analysis using the X-ray diffraction phenomenon, a crystal (single crystal) in which molecular arrangements defined by a unit cell are uniformly arranged as a whole is required. However, in the crystal growth process, a specific growth surface of one single crystal becomes the growth start point of a new single crystal, and apparently one crystal (twin crystal) and two or more single crystals cannot be separated. In some cases, a twining may occur, or a polycrystalline structure in which a plurality of single crystals adhere to each other may occur. If the single crystal can be separated, structural analysis may be possible for the crystal having a polycrystalline structure. However, since the crystal is small or physically damaged, it is generally not suitable for structural analysis.

  Usually, as a method for solving the above problems, for example, a surfactant, an organic solvent such as dioxane, or a saccharide such as glycerol is added, or a method for modifying a residue on the surface of a protein molecule is known. It was done. However, with regard to the crystal of the present invention, the surfactant, organic solvent, and the like have little effect and it is difficult to produce a single crystal. As a result of intensive studies, the present inventors have found that the addition of a diamine compound is effective in promoting the growth of a single crystal of the present invention, and have obtained the crystal of the present invention.

  The diamine compound is usually used as a chelating agent in order to stabilize a protein in crystallization. However, the diamine compound is used to suppress twinning or polycrystalline structure and promote single crystal growth. That the present inventors have found for the first time.

  The crystal of the present invention is prepared by, for example, using a γGCS solution in which purified E. coli γGCS and a compound represented by the following structural formula (8) are incubated in the presence of magnesium / ATP and a diamine compound is added, It can crystallize by performing a diffusion method. The purification method of the E. coli γGCS is not particularly limited, and can be purified by a conventionally known method.

  Examples of the diamine compound added to the γGCS solution include 1,6-diaminohexane and 1,8-diaminooctane, and the concentration of the diamine compound added is, for example, 0.3 to 0.3 as the initial concentration of the γGCS solution. It is 8% (W / V), preferably 0.5 to 0.7% (W / V), and more preferably 0.5% (W / V). The pH of the γGCS solution at the time of adding the diamine compound is 8.0 to 9.5. If the pH exceeds 9.5, the enzyme is denatured, and if the pH is less than 8.0, no crystals are formed. The pH is preferably 8.0 to 9.0, and more preferably 8.0 to 8.5.

  When the compound selected from the drug candidate compounds by the screening method of the present invention undergoes pharmacological experiments regarding effects on human bodies, side effects, etc., and is pharmaceutically acceptable, parasites can be obtained by a conventionally known method. It can be used as a therapeutic / preventive drug for infectious diseases such as

  The present invention will be further described below with reference to examples.

(Crystallization of γGCS)
The expression / purification of γGCS was performed as follows. First, after culturing cells of Escherichia coli (JM109), the cell membrane was broken by an ultrasonic disruption method or the like to prepare a crude cell extract. Ammonium sulfate is added to the obtained crude cell extract so as to be 45-50% saturated, and ammonium sulfate is precipitated. Then, the supernatant fraction obtained by centrifugation is used for hydrophobic column chromatography and ATP agarose affinity column chromatography. Separation by DEAE or QAE anion exchange column chromatography was performed in this order to obtain a purified enzyme preparation.

Next, the purified γGCS and the compound represented by the structural formula (8) were incubated in the presence of magnesium · ATP at 25 ° C. until the activity was completely stopped. This complex was crystallized at 20 ° C. against a buffer of 20 mM Tris-HCl, pH 7.5 containing 15% (W / V) PEG 2000 MME and 250 mM MgCl ₂ by a sitting drop vapor diffusion method. In addition, 0.5% (W / V) diaminooctane was added to the γGCS solution in order to prevent twinning. The obtained crystals were instantaneously immersed in an antifreeze solution containing 25% PEG400 and then held in liquid nitrogen directly until data collection.

(Data collection)
X-ray diffraction data of the crystals were collected at 100K using a MAR CCD detector at SPring-8 beamline BL41XU. The intensity data was processed using a combination of the MOSFLM program and the CCP4 program (manufactured by Collaborative Computational Project) or using the product name: CrystalClear software (manufactured by Rigaku / MSC, Inc.). The results are shown in Table 3 below. As shown in Table 3 below, a co-crystal having a resolution of 2.1 mm and capable of obtaining three-dimensional structure analysis data with higher resolution than that of the conventional crystal was obtained.

(Table 3)
Data collection statistics
Space group P2 ₁
Unit cell parameters a = 70.45 Å, b = 97.36 Å
c = 102.18 mm, β = 109.63 °
No. of monomers / asymmetric unit 2
Wavelength (Å) 1.0000
Resolution (Å) 48.8-2.10 (2.21-2.10)
Total No. of reflections 280,959
No. of unique reflections 73,644
Completeness (%) 97.2 (96.2)
Redundancy 3.8 (3.8)
I / σ 13.3 (3.2)
R _merge (= Σ _h Σ _i | I _{h, I-} <I _h > | / Σ _h Σ _i | I _{h, i} |) 0.080 (0.371)

(ΓGCS multiple alignment)
Multiple alignment of cysteine binding sites of γGCS (FIG. 2) was performed as follows.
(1) First, normal multiple alignment was performed on relatively closely related γGCS.
(2) Next, a glutamic acid binding site, a nucleotide binding site, and an Mg binding site that are highly conservative and easy to distinguish were identified. Then, the remaining part of the sequence A includes a cysteine-binding site.
(3) The crystal structure obtained in Example 1 was analyzed, and the cysteine binding site was composed of an N-terminal variable arm consisting of 40 amino acid residues, a central variable arm consisting of 59 residues, and a switch loop consisting of 10 residues. The three-dimensional structure information was obtained and used as the basic structure of the cysteine binding site. Then, in the sequence A, the sequence A was applied to the basic structure, assuming that a portion with a large variation was a location away from the variable arm and a location with high storage stability was in the variable arm.
(4) As a result, a region consisting of 40 amino acid residues (corresponding to the N-terminal variable arm) and a region consisting of 73 amino acid residues (corresponding to the central variable arm and the switch loop) are cut out from the sequence A, and sequentially aligned. Then, the alignment shown in FIG. 2 was obtained by selecting the combination having the highest homology.

(Purification of non-human recombinant γGCS)
As a non-human γGCS, an R132N mutant (R132N mutant γGCS) of trypanosome-blues E. coli γGCS was constructed, and host E. coli (JM109) was transformed to produce an R132N mutant γGCS-producing strain. The R132N mutant γGCS-producing strain was cultured, and the cells were collected, sonicated, and then ultracentrifuged. Using the supernatant fraction, ion exchange chromatography with DEAE-Toyopearl (manufactured by Tosoh Corporation) and affinity column chromatography with ATP agarose were performed to obtain a purified enzyme preparation of R132N mutant γGCS.

(Sensitivity of non-human recombinant γGCS to sulfoximine type inhibitors)
γGCS synthesizes γ-glutamylcysteine from L-Glu and L-Cys by consuming ATP in the presence of Mg ²⁺ (see FIG. 3). By measuring the amount of ADP produced at this time, the activity of γGCS can be measured. In addition, the amount of ADP can be determined by applying NADH to phosphoenolpyruvate (PEP), pyruvate kinase (PK), lactate dehydrogenase (LDH) and NADH by applying a coupled enzyme method. NAD ⁺ production amount). The molar extinction coefficient of NADH at a wavelength of 340 nm is ε = 6220 M ⁻¹ · cm ⁻¹ .

Using the above-mentioned γGCS activity measurement system, the activities of R132N mutant γGCS and wild type γGCS when sulfoximine type inhibitors (substrate transition state analogs) were added at various concentrations were measured. Specifically, a reaction solution containing a sulfoxyimine type inhibitor (25 mM L-Cys, 15 mM L-Glu, 1 mM ATP, 10 mM MgSO ₄ , 0.24 mM NADH, 1 mM PEP, 10 unit PK, 25 unit LDH, 0.1 M KCl, The reaction was started by adding 50 μl of 3.0 μg / ml wild-type γGCS or 120 μg / ml R132N mutant γGCS to 0.1 M Tris-HCl pH 7.5), and the change with time in absorbance at a wavelength of 340 nm was measured for 30 minutes. . In addition, enzyme 1 unit shows the amount of enzyme which consumes 1 micromol substrate per minute at 37 degreeC. Moreover, BSO of the following formula (9) and an inhibitor of the following formula (10) were used as the sulfoximine type inhibitors. The results are shown in FIG. 4 and Table 4.

In the above formula (10), R is —CH ₂ OH, —CH ₂ CH ₃ or —CH ₂ CH ₂ CH ₃ .

(Table 4)
Transition state inhibition constant Ki (μM) Rate constant kon (M ⁻¹ s ⁻¹ )
Analog wild type R132N wild type R132N
R = CH ₂ OH 8.9 39 10 1.6
BSO 49> 1000 4.9 <0.001
R = Et 0.099> 1000 1400 <0.001
R = n-Pr 0.59> 1000 220 <0.001

FIG. 4 shows the measurement of ADP production over time for wild-type γGCS and R132N mutant γGCS using BSO of the above formula (9) and R═CH ₂ OH of the above formula (10) as sulfoximine type inhibitors. It is a graph. Table 4 shows the results of calculating the rate constant kon and the inhibition constant Ki for the wild-type γGCS and R132N mutant γGCS of the four types of sulfoximine inhibitors. Here, as described above, the inhibition constant Ki was calculated as Ki = [E] · [I] / [EI *].

As shown in FIG. 4 and Table 4, R132N mutant γGCS is used for BSO which has been conventionally used as an inhibitor and sulfoxyimine type inhibitors having a hydrophobic functional group (R = Et, n-Pr). Inhibition constant Ki was only 1000 μM or more, indicating very weak sensitivity. On the other hand, an inhibitor having a polar functional group (R = CH ₂ OH) showed stronger inhibition against R132N mutant γGCS than to inhibit BSO against wild-type γGCS. Also, inhibitors having a functional group (R = CH ₂ OH) with a polarity relative GanmaGCS, the degree of inhibition exhibited over time larger inhibitory activity of slow binding inhibition of symptoms (see Fig. 4). These results show that after the hydroxyl group of R = CH ₂ OH and the 132nd asparagine (N) of the R132N mutant γGCS form a hydrogen bond to form an enzyme / inhibitor complex, the inhibitor is bound by ATP. Phosphorylation suggests that quasi-irreversible inactivation of the enzyme is occurring.

  The molecular design of an inhibitor of γGCS that specifically selects and inhibits non-human γGCS in which the amino acid residue corresponding to the 132nd arginine of γGCS of Escherichia coli is substituted with asparagine or glutamine is L-cysteine. It was suggested that it is preferable to design so as to include a functional group capable of forming a hydrogen bond with the asparagine or glutamine considered to form a side chain recognition site.

  As described above, the present invention is useful for developing inhibitors specific to non-human γGCS including γGCS of parasites such as Trypanosoma blues and P. falciparum, for example, African sleep It is useful in the field of treatment / prevention drugs and treatment / prevention methods for diseases and parasitic infections such as falciparum malaria.

FIG. 1A shows an example of the three-dimensional structure of a complex containing E. coli γGCS, Mg, ADP and a substrate transition state analog, and FIG. 1B shows an example of the two-dimensional structure of E. coli γGCS. FIG. 2 shows an example of the result of multiple alignment of the N-terminal variable arm and the central variable arm constituting the L-cysteine binding site of γGCS. FIG. 3 is a diagram illustrating a glutathione biosynthesis system. FIG. 4 is a graph showing an example of the results of measuring the sensitivity of wild type and mutant γGCS to sulfoxyimine type inhibitors.

Claims (11)

An amino acid residue located at the L-cysteine binding site of γGCS and corresponding to the amino acid sequence at position 132 when aligned with the amino acid sequence of SEQ ID NO: 1 is an amino acid residue other than arginine A non-human recombinant γGCS as described in (i) or (ii) below.
(I) an amino acid sequence in which the 132nd arginine of E. coli γGCS amino acid sequence (SEQ ID NO: 1) is substituted with asparagine or glutamine;
An amino acid sequence in which the 131st arginine of Vibrio cholerae γGCS amino acid sequence (SEQ ID NO: 2) is substituted with asparagine or glutamine,
An amino acid sequence in which the 134th arginine of Pseudomonas aeruginosa γGCS amino acid sequence (SEQ ID NO: 3) is substituted with asparagine or glutamine;
An amino acid sequence in which the 132rd arginine of Salmonella γGCS amino acid sequence (SEQ ID NO: 4) is substituted with asparagine or glutamine;
An amino acid sequence in which the 132rd arginine of the Bufnella γGCS amino acid sequence (SEQ ID NO: 5) is substituted with asparagine or glutamine;
An amino acid sequence in which the 191st arginine of the human γGCS amino acid sequence (SEQ ID NO: 6) is substituted with asparagine or glutamine;
An amino acid sequence in which the 191st arginine of the rat γGCS amino acid sequence (SEQ ID NO: 7) is substituted with asparagine or glutamine;
An amino acid sequence in which the 191st arginine of the mouse γGCS amino acid sequence (SEQ ID NO: 8) is substituted with asparagine or glutamine;
The amino acid sequence in which the 112th arginine of the mycoplasma γGCS amino acid sequence (SEQ ID NO: 9) is substituted with asparagine or glutamine, and the 185th arginine of the trypanosome glue γGCS amino acid sequence (SEQ ID NO: 10) is asparagine or glutamine. A non-human recombinant γGCS comprising an amino acid sequence selected from the group consisting of amino acid sequences substituted with
(Ii) In the amino acid residue other than the amino acid residue substituted with asparagine or glutamine in the amino acid sequence of (i) above, one or several amino acid residues consist of an amino acid sequence deleted, substituted or added. And a non-human recombinant γGCS having γ-glutamylcysteine synthetase activity. An amino acid residue located at the L-cysteine binding site of γGCS, wherein the amino acid residue corresponding to position 132 of the amino acid sequence is arginine when aligned with the amino acid sequence of SEQ ID NO: 1. A human-type recombinant γGCS comprising the amino acid sequence described in (iii) or (iv) below:
(Iii) an amino acid sequence obtained by substituting arginine for the 185th asparagine of trypanosoma blues γGCS (SEQ ID NO: 11), or
A human-type recombinant γGCS consisting of an amino acid sequence in which the 473th glutamine of P. falciparum γGCS (SEQ ID NO: 12) is substituted with arginine.
(Iv) an amino acid residue other than the amino acid residue substituted with arginine in the amino acid sequence of (iii) above, wherein one or several amino acid residues are deleted, substituted or added, and , A human recombinant γGCS having γ-glutamylcysteine synthetase activity. Polynucleotide encoding a human recombinant γGCS according to a non-human recombinant γGCS or claim 2 of claim 1. A vector comprising the polynucleotide according to claim 3 . An organism transformed with the vector according to claim 4 (excluding humans) or a cell thereof. A method for screening an inhibitor of γ-glutamylcysteine synthetase (γGCS),
Preparing the non-human recombinant γGCS according to claim 1 and the human recombinant γGCS according to claim 2 ,
A drug candidate compound prepared in advance is brought into contact with each of the human type and non-human type γGCS, and the strength of the binding of the compound to both γGCSs is evaluated, respectively.
Screening method characterized by comprising the step of selecting the drug candidate compound than the human GanmaGCS binds strongly by the non-human GanmaGCS. The inhibitor to be screened is an inhibitor of Trypanosoma brucei γGCS, and the non-human recombinant γGCS is the 132nd amino acid sequence when aligned with the amino acid sequence of SEQ ID NO: 1. The method according to claim 6 , wherein the amino acid residue corresponding to is γGCS, which is asparagine. The inhibitor to be screened is an inhibitor of Plasmodium falciparum γGCS, and the non-human recombinant γGCS is the first of the amino acid sequence when aligned with the amino acid sequence of SEQ ID NO: 1 . The method according to claim 6 , wherein the amino acid residue corresponding to position 132 is γGCS, which is glutamine. The screening method according to any one of claims 6 to 8 , wherein a compound having a chemical structure represented by the following general formula (2) is used as the drug candidate compound.

In the above formula (2), X represents —H, —CH ₃ , —CH ₂ CH ₃ , —CH ₂ OH, —CH ₂ SH, —CH ₂ NH ₂ , —CH ₂ NHCH ₃ , —CH ₂ N ( _{CH 3) 2, -CF 3,} -CH 2 Br, -CH 2 Cl, -CHCl 2, -CN, -NC, -CH 2 CN, -CH 2 NC, -CH 2 CH 2 CH 3, -CH 2 _{CH 2 OH, -CH 2 CH 2} SH, -CH 2 CH 2 Cl, -CH 2 CH 2 Br, -CH 2 CF 3, -COOH, -CH 2 COOH, -COCH 3, -COCH 3, -PO 3 H, —CH ₂ PO ₃ H, —P (CH ₃ ) O ₂ H, —SO ₃ H, —SO ₂ H, —NO ₂ , —C≡CH, —CH═CH ₂ , —CONH ₂ , —CH ₂ COCNH _2, and a substituent selected from the group consisting of -CH ₂ CH ₂ NH _2, Y is, -H, -OH, -COOH, _{NH 2, -COOCH 3, -CONH 2} , -CN, -SO 3 H and, a substituent selected from the group consisting of -NO _2. The screening method according to any one of claims 6 to 9 , wherein the method for evaluating the strength of binding is an assay method based on slow-binding kinetics. A crystal used for designing a drug candidate compound in the screening method according to any one of claims 6 to 10 , wherein the crystal is a co-crystal of γGCS comprising the amino acid sequence of SEQ ID NO: 1 and a compound represented by the following structural formula: , crystal space group, Ri P2 ₁ der, the unit cell, a = 70.45Å, b = 97.36Å , c = 102.18Å, characterized beta = 109.63 ° der Rukoto.

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