D-serine transport regulator and screening method thereof, and screening method for D-serine transporter proteins

Abstract

The present invention provides a D-serine transport modifier which is characterized by controlling the transport of D-serine into and out of cells by a D-serine transporter protein, a pharmaceutical composition which comprises the same as an active component and treats or prevents diseases relating to an increase or decrease in the amount of D-serine, and a screening method of substances that control the transport of D-serine. The present invention also provides a screening method of a D-serine transporter protein.

Description

[Technical Field]

[0001] The present invention relates to a D-serine transport regulator, a pharmaceutical composition for treating or preventing diseases associated with an increase or decrease in D-serine levels, a method for treating or preventing diseases associated with an increase or decrease in D-serine levels, and a method for screening substances that control D-serine transport. The present invention also relates to a method for screening D-serine transporter proteins. [Background technology]

[0002] D-amino acids, which were previously thought not to exist in mammalian bodies, are now being shown to be present in various tissues and to play a role in physiological functions. D-serine, one of the D-amino acids, has been shown to be a potential biomarker that reflects kidney function and kidney disease (Non-patent documents 1-3).

[0003] However, in the process of developing D-serine as a kidney disease marker, the transport system for D-serine in the kidney was not clearly understood. To understand the physiological significance of D-serine, it was necessary to identify the transporter molecules involved in the mechanism of D-serine transport into and out of cells, and this was strongly desired.

[0004] ASCT1(SLC1A4) (Non-Patent Documents 4 and 5), ASCT2(SLC1A5) (Non-Patent Document 5), Asc1(SLC7A10) (Non-Patent Documents 6 and 7), PAT1(SLC36A1) (Non-Patent Document 8), and ATB 0,+ (SLC6A14) (Non-Patent Document 9) is known to play a role as a D-serine membrane transporter protein. However, it has not been clarified whether these proteins play a role as D-serine membrane transporters in the kidney. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sasabe J., et al., Ischemic acute kidney injury perturbs homeostasis of serine enantiomers in the body fluid in mice: early detection of renal dysfunction using the ratio of serine enantiomers. PLoS One. 2014 Jan 29; 9(1):e86504. [Non-Patent Document 2] Silbernagl S., et al., D-Serine is reabsorbed in rat renal pars recta. Am J Physiol. 1999 Jun; 276(6):F857-63. [Non-Patent Document 3] Hesaka A., et al., D-Serine reflects kidney function and diseases. Sci Rep. 2019 Mar 25;9(1):5104. [Non-Patent Document 4] Kaplan E. et al., ASCT1 (Slc1a4) transporter is a physiologic regulator of brain d-serine and neurodevelopment. Proc Natl Acad Sci US A. 2018 Sep 18;115(38):9628-9633. [Non-Patent Document 5] Foster AC., et al., D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1 / SLC1A4 and ASCT2 / SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures. PLoS One. 2016 Jun 7;11(6):e0156551. [Non-Patent Document 6] Kasai Y., et al., Transport systems of serine at the brain barriers and in brain parenchymal cells. J Neurochem. 2011 Jul;118(2):304-13.

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0006] There is a need for the development of drugs to treat or prevent diseases associated with elevated or decreased D-serine levels, such as kidney disease. To achieve this, it is necessary to identify novel D-serine transporter proteins capable of regulating the amount of D-serine in cells, tissues, organs, or body fluids, and screening methods for such proteins are urgently needed. [Means for solving the problem]

[0007] As a result of diligent research by the present inventors, we have discovered a D-serine transporter protein that can regulate the amount of D-serine in cells, tissues, organs, or body fluids. We have also found that by using such a D-serine transporter protein or a substance that acts on it as a D-serine transport regulator, it is possible to treat or prevent diseases associated with elevated or decreased D-serine levels (e.g., kidney disease). Furthermore, we have developed a method for easily screening novel D-serine transporter proteins that can regulate the amount of D-serine in cells, tissues, organs, or body fluids, as well as novel substances that act on D-serine transporter proteins, leading to the present invention. In other words, the present invention encompasses the following inventions.

[0008] [1] A D-serine transport regulator characterized by controlling the transport of D-serine into and out of cells by D-serine transporter proteins. [2] The D-serine transport regulator according to item 1, wherein the D-serine transporter protein is selected from one or more of the first group of D-serine transporter proteins consisting of the SMCT family, GLUT5, CAT1, THTR2, and SNAT2. [3] The D-serine transporter proteins include ASCT family Asc1, PAT1 and ATB 0,+ A D-serine transport regulator according to item 2, further comprising one or more D-serine transporter proteins selected from the group of second D-serine transporter proteins consisting of the above. [4] A D-serine transport regulator according to any one of items 1 to 3, characterized by regulating the amount of D-serine in cells, tissues, organs, or body fluids. [5] A D-serine transport regulator according to any one of items 1 to 3, characterized by regulating the amount of D-serine in the blood and / or urine. [6] A D-serine transport regulator according to any one of items 1 to 5, characterized by inhibiting the transport of D-serine to cells by acting on the D-serine transporter protein. [7] D-serine transport regulators as described in item 6, selected from the group consisting of antisense RNA or DNA molecules, RNAi-inducible nucleic acids, microRNAs (miRNAs), ribozymes, genome-edited nucleic acids and their expression vectors, small molecule compounds, aptamers, antibodies, antibody fragments, and combinations thereof. [8] A D-serine transport regulator according to item 6, which is a substrate or inhibitor of the D-serine transporter protein. [9] Ibuprofen, fenoprofen, ketoprofen, probenecid, acetylsalicylic acid, naproxen, pyroglutamic acid, phenoxyacetic acid, acetic acid, propionic acid, butyric acid, L-lactic acid, D-lactic acid, pyruvate, nicotinic acid, acetoacetic acid, β-D-hydroxybutyric acid, β-L-hydroxybutyric acid, γ-hydroxybutyric acid, α-ketoisocaproic acid, benzoic acid, salicylic acid, 5-aminosalicylic acid, 2,4-dichloro Phenoxyacetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPA), 2-chlorophenoxyacetic acid (2-CPA), 2,3-dichlorophenoxyacetic acid, 3,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, N-(4-methanesulfonyl-2-nitrophenyl)-2H-1,3-benzodioxol-5-amine (MSNBA), fructose, N-ethylmaleimide (NEM), A D-serine transport regulator according to item 6, selected from one or more of the group of first substrates or inhibitors of the D-serine transporter protein, comprising N-amino-L-arginine, N-amino-L-homoarginine, L-arginine, L-histidine, L-lysine, L-ornithine, metformin, chloroquine, 2,4-diaminopyrimidine, fedratinib, AZD1480, cerdulatinib, thiamine, methyl-amino-isobutyric acid (MeAIB), γ-glutamyl-p-nitroanilide (GPNA), 2-amino-4-bis(aliloxybenzyl)aminobutanoic acid (AABA), L-alanine, L-methionine, L-proline, L-serine, L-asparagine, L-glutamine, L-histidine, glycine and its derivatives, and pharmaceutically acceptable salts thereof.

[10] A D-serine transport modifier according to item 9, further comprising one or more substances selected from the group of second substrates or inhibitors of the D-serine transporter protein, consisting of phenylglycine analogs, benzylserine, benzylcysteine, S-benzyl-L-cystine, L-γ-glutamyl-p-nitroanilide, L-serine, L-threonine, L-methionine, L-alanine, L-cysteine, L-glutamine, D-alanine, phenylglycine analogs, alanine analogs, L-serine, L-alanine, L-cysteine, glycine, L-threonine, taurine, GABA, tryptophan, tryptamine derivatives, 5-hydroxy-L-tryptophan, serotonin, indole-3-propionic acid, α-methyl-DL-tryptophan and its derivatives, and pharmaceutically acceptable salts thereof.

[11] A pharmaceutical composition for treating or preventing diseases associated with elevated levels of D-serine in cells, tissues, organs or body fluids, comprising as an active ingredient any one of items 6 to 10.

[12] The pharmaceutical composition according to item 11, wherein the disease associated with the increase in D-serine levels is kidney disease.

[13] A method for treating or preventing a disease associated with elevated levels of D-serine in cells, tissues, organs or body fluids, comprising administering a D-serine transport modulator described in any one of items 6 to 10 to a subject in need thereof.

[14] The method of item 13, wherein the disease associated with the increase in D-serine levels is kidney disease.

[15] A D-serine transport regulator according to any one of items 1 to 5, characterized in that it promotes the transport of D-serine to cells by acting on the D-serine transporter protein.

[16] A D-serine transport regulator according to item 15, selected from the group consisting of the D-serine transporter protein, a derivative thereof or a vector expressing a part thereof, a small molecule compound, an aptamer, an antibody, an antibody fragment, and combinations thereof.

[17] A D-serine transport regulator according to item 15, selected from the group consisting of diclofenac, curcumin, activin A, and SMCT family, GLUT5, CAT1, THTR2, SNAT2, and PDZK1 expression vectors.

[18] A pharmaceutical composition for treating or preventing diseases associated with a decrease in intracellular, intratissue, intraorganic or intrafluid levels of D-serine, comprising as an active ingredient any one of items 15 to 17.

[19] The pharmaceutical composition according to item 18, wherein the disease associated with the decrease in D-serine levels is kidney disease.

[20] A method for treating or preventing a disease associated with a decrease in intracellular, intratissue, intraorganic or intrafluid levels of D-serine, comprising administering a D-serine transport modulator described in any one of items 15-17 to a subject in need thereof.

[21] The method according to item 20, wherein the disease associated with the decrease in D-serine levels is kidney disease.

[22] A method for screening substances that control the transport of D-serine into and out of cells by D-serine transporter proteins, A process in which a candidate substance and D-serine are applied to cells expressing D-serine transporter proteins, and the degree of D-serine transport into and out of the cell is evaluated using the expression of cytotoxicity as an indicator. Methods that include...

[23] The method according to item 22, wherein the D-serine transporter protein is selected from one or more of the first group of D-serine transporter proteins consisting of the SMCT family, GLUT5, CAT1, THTR2, and SNAT2.

[24] The D-serine transporter proteins are ASCT family Asc1, PAT1 and ATB 0,+ The method according to item 23, further comprising one or more D-serine transporter proteins selected from the group of second D-serine transporter proteins consisting of the following.

[25] The method according to any one of items 22 to 24, wherein the cells are cells obtained by introducing a vector expressing the D-serine transporter protein from an external source.

[26] The method according to any one of items 22 to 24, for selecting a substance that inhibits the transport of D-serine into cells by acting on the D-serine transporter protein, and for screening for a substance that treats or prevents a disease associated with elevated levels of D-serine in cells, tissues, organs or body fluids.

[27] The method according to item 26, wherein the disease associated with the increase in D-serine levels is kidney disease.

[28] The method according to any one of items 22-24, for selecting a substance that promotes the transport of D-serine into cells by acting on the D-serine transporter protein, and for screening for a substance that treats or prevents a disease associated with a decrease in the amount of D-serine in cells, tissues, organs or body fluids.

[29] The method according to item 28, wherein the disease associated with the decrease in D-serine levels is kidney disease.

[30] A screening method for D-serine transporter proteins, using the transport of D-serine into cells as an indicator.

[31] The method according to item 30, wherein the expression of cytotoxicity caused by the addition of D-serine is used as an indicator of the transport of D-serine to the cells.

[32] The method according to item 30 or 31, wherein the cells are cells expressing a candidate transporter protein.

[33] The method according to item 32, wherein the cells are cells obtained by introducing a vector expressing the candidate transporter protein.

[34] The method according to item 33, wherein the vector is selected from the group consisting of plasmid vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, and viral vectors. [Effects of the Invention]

[0009] According to the present invention, it becomes possible to regulate the amount of D-serine in cells, tissues, organs, or body fluids, and consequently, it becomes possible to treat or prevent diseases associated with an increase or decrease in D-serine levels, such as kidney disease. Furthermore, according to the present invention, it becomes possible to discover novel D-serine transporter proteins that can regulate the amount of D-serine in cells, tissues, organs, or body fluids, or novel substances that act on D-serine transporter proteins. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows that D-serine transport in mouse brush border membrane vesicles (BBMVs) is primarily Na+-dependent. The time course of D-[3H]serine uptake activity at 10 μM (A) or 50 μM (B) in BBMVs was measured in the presence (Na+) and absence (K+). *P<0.05. [Figure 2] Figure 2 shows that D-serine transport in BBMV was inhibited primarily by ASCT2 and SMCT inhibitors. The results of measuring D-[3H]serine transport (10 μM) activity in the presence and absence of 1 mM nicotinic acid or 2 mM L-threonine (L-Thr) are shown. *P<0.05, NS: no significant difference. [Figure 3]Figure 3 shows that human SMCT1 and hSMCT2 transport D-serine. Stable cell lines of FlpIn293TR-hSLC5A8-3xFLAG(hSMCT1) or FlpIn293TR-hSLC5A12-3xFLAG(hSMCT2) were prepared. (A) Flp-In TREx 293 cells were knocked down with ASCT2 siRNA, and expression was confirmed by Western blotting using an anti-ASCT2 antibody. (B) Two days before the uptake experiment, doxycycline was added to hSMCT1 and SMCT2 cells to induce expression. This expression was confirmed by Western blotting using an anti-FLAG antibody. (C) The time course of 100 μM D-[3H]serine uptake was measured in ASCT2-endogenous hSMCT1 and SMCT2 stable cell lines. (D) Measurements were taken in hSMCT1 and SMCT2 stable cell lines using ASCT2 knockdown. Graphs (E) and (D) show the difference between the observed values ​​in hSMCT1 and SMCT2 stable cell lines and the observed values ​​in mock cells. *P<0.05, **P<0.01, NS: No significant difference. [Figure 4] Figure 4 shows that D-serine transport mediated by the SMCT transporter was inhibited by NSAIDs. *P<0.05, **P<0.01. [Figure 5] Figure 5 shows that D-serine transport activity in mouse brush border membrane vesicles (BBMVs) was inhibited by the SMCTs inhibitor ibuprofen. **P<0.01. [Figure 6] Figure 6 shows that D-serine inhibited the proliferation of Flp-In TREx293 cells. Flp-In TREx293 cells were treated with L- or D-serine for 2 days, and cell proliferation was measured by the XTT assay. The same data are shown in (A) linear curve plot and (B) semi-logarithmic plot. *P<0.05 [Figure 7] Figure 7 shows that D-serine transport was driven by the cell line's Na+-dependent transporter. Time-dependent changes in 10 μM D-[3H]serine uptake activity were measured using (A) HEK293 cells and (B) Flp-In TREx 293 cell line. **P<0.01. [Figure 8] Figure 8 shows that D-serine transport was relatively inhibited by ASCT2 inhibitors. **P<0.01, NS: No significant difference. [Figure 9] Figure 9 shows that the ASCT2 transporter is expressed in Flp-In TREx 293 cells, and its knockdown (KD) reduces the toxicity of D-serine. (A) Western blotting shows that the ASCT2 transporter is endogenously expressed in Flp-In TREx 293 cells. (B) Flp-In TREx 293 control cells and ASCT2 knockdown cells were treated with L- or D-serine for 2 days, and cell proliferation was measured by XTT. *P<0.05. [Figure 10] Figure 10 shows the toxicity tests of D-serine using transient expression systems of SMCT1 and SMCT2. The toxicity tests of D-serine were performed in HEK293 cells transiently transfected with (A) pCMV14-hSLC5A8-3xFLAG (SMCT1) or (B) pCMV14-hSLC5A12-3xFLAG (SMCT2). *P<0.05. [Figure 11-1] Figure 11 shows the construction of hSMCT stable cell lines. (A) Vector map of pCDNA5-hSLC5A8-3xFLAG for constructing the hSMCT1-3xFLAG stable cell line. (B) Vector map of pCDNA5-hSLC5A12-3xFLAG for constructing the hSMCT2-3xFLAG stable cell line. [Figure 11-2] Figure 11 shows the construction of stable hSMCT cell lines. (C) hSMCT1-3xFLAG (arrowhead) and hSMCT2-3xFLAG (arrow) were subjected to Western blotting using an anti-FLAG antibody. [Figure 12] Figure 12 shows that SMCT2 enhanced the D-serine-induced inhibition of cell proliferation. The cell proliferation effect of serine treatment was investigated in FlpIn293TR-Mock (Mock) or FlpIn293TR-hSLC5A12-3xFLAG(SMCT2) stable cell lines. [Figure 13]Figure 13 shows that ibuprofen reduced D-serine sensitivity in SMCT2 stable cell lines. *P<0.05, NS: no significant difference. [Figure 14] Figure 14 shows that SMCT1 increased D-serine sensitivity, and ibuprofen counteracted this increased D-serine sensitivity. *P<0.05, NS: No significant difference. [Figure 15] Figure 15 shows the proteome volcano plot of the renal brush border membrane fraction from renal ischemia-reperfusion injury (IRI) mice. The volcano plot shows log2 (Fold Change) (8h / 0h) plotted against the statistical significance P-value (-log 10) from the data of all identified proteins (A) or transporters (B). The known D-serine transporter ASCT2 (SLC1A5), which shows a log2 (Fold Change) of 0.54 and a -log10 P-value of 0.80, was used as the cutoff value (arrow). [Figure 16] Figure 16 shows D-serine-induced cytotoxicity in HEK293 cells transfected with candidate D-serine transporters. HEK293 cells were transfected with each of the candidate transporters shown in the figure. These cells were treated with D-serine at a concentration of 15 mM (A) or 25 mM (B) for 2 days. Cytotoxicity was evaluated by the XTT assay. The D-serine toxicity effect in each transfected cell was normalized to that of cells without D-serine treatment and compared to the effect of Mock at the same D-serine concentration. Significant reductions were calculated by t-tests. *p<0.05; **p<0.01. [Figure 17] Figure 17 shows the results of identifying SNAT2 as a D-serine transporter in ASCT2 knockout HAP1 cells. (A) D-serine transport was measured in wild-type HAP1 cells and ASCT2 knockout HAP1 cells. D-serine transport was reduced by approximately 30% in ASCT2 knockout cells. (B) D-serine transport was measured in wild-type HAP1 cells and ASCT2 knockout HAP1 cells in the presence and absence of SNAT1 and the SNAT2 inhibitor MeAIB. [Figure 18] Figure 18 shows D-serine transport in ASCT2 knockout HAP1 cells stably expressing candidate cDNAs. A stable cell line expressing the cDNA clones mentioned was constructed using ASCT2-knockout HAP1 cells. The ASCT2 stable cell line was used as a positive control. [3H]D-serine uptake was measured for 10 minutes in the presence of MeAIB, and SNAT1 and SNAT2 activity were subtracted from the background. Uptake values ​​were normalized using mock values. As a result, SMCT1 showed high D-serine uptake. *p<0.05; **p<0.01. [Figure 19] Figure 19 shows the inhibition of [3H]D-serine transport by GABA. (A) Results of measuring [3H]D-serine transport in HEK293 cells are shown. It was shown that the ASCT2 substrate and GABA inhibited [3H]D-serine uptake. (B) [3H]D-serine transport was measured in HEK293 cells transfected with ASCT2. The inhibitory effects of L-serine and GABA were investigated after addition. Both GABA and L-serine significantly inhibited [3H]D-serine transport. [Modes for carrying out the invention]

[0011] The following describes embodiments for carrying out the present invention, but the technical scope of the present invention is not limited to the embodiments described below.

[0012] In this specification, terms such as "first," "second," etc., are used solely to distinguish one element from another and have no further implications. Therefore, for example, the first element may be referred to as the second element, and similarly the second element as the first element, and this will not deviate from the scope of the present invention.

[0013] One embodiment of the present invention provides a D-serine transport regulator characterized by controlling the transport of D-serine into and out of cells by D-serine transporter proteins.

[0014] "D-serine" is an optical isomer of L-serine, an amino acid that makes up proteins. In this specification, "D-serine transporter protein" (also called "D-serine transporter") refers to a general term for proteins that span biological membranes and transport D-serine across the membrane.

[0015] In this specification, "transport of D-serine into and out of cells" means a concept that includes the transport of D-serine from inside to outside the cell, from outside to inside the cell, or between multiple cells, via D-serine transporter proteins.

[0016] In one embodiment, the present invention may be a D-serine transport regulator that controls the transport of D-serine into and out of cells by one or more D-serine transporter proteins selected from the group of D-serine transporter proteins consisting of the SMCT family, GLUT5, CAT1, THTR2, and SNAT2 (referred to as the "first group of D-serine transporter proteins" as appropriate). In this specification, "SMCT family" refers to the SMCT protein family, including SMCT1 and SMCT2.

[0017] SMCT1 is a sodium-coupled monocarboxylate transporter 1 protein encoded by the human SLC5A8 gene. The mRNA and amino acid sequences of human SMCT1 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_145913 (SEQ ID NO: 1) and NP_666018 (SEQ ID NO: 2), respectively, and can be used in this invention.

[0018] SMCT2 is a sodium-coupled monocarboxylate transporter 2 protein encoded by the human SLC5A12 gene. The mRNA and amino acid sequences of human SMCT2 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_178498 (SEQ ID NO: 3) and NP_848593 (SEQ ID NO: 4), respectively, and can be used in this invention.

[0019] GLUT5 is a glucose transporter 5 protein encoded by the human SLC2A5 gene. The mRNA and amino acid sequences of human GLUT5 are provided, for example, in the GenBank database and the GenPept database as acceptance numbers NM_003039 (SEQ ID NO: 5) and NP_003030 (SEQ ID NO: 6), respectively, and can be used in this invention.

[0020] CAT1 is a Cationic amino acid transporter 1 protein encoded by the SLC7A1 gene. The mRNA and amino acid sequences of human CAT1 are provided, for example, in the GenBank database and the GenPept database as access numbers NM_003045 (SEQ ID NO: 7) and NP_003036 (SEQ ID NO: 8), respectively, and can be used in this invention.

[0021] THTR2 is a Thiamine transporter 2 protein encoded by the SLC19A3 gene. The mRNA and amino acid sequences of human THTR2 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_025243 (SEQ ID NO: 9) and NP_079519 (SEQ ID NO: 10), respectively, and can be used in this invention.

[0022] SNAT2 is a sodium-coupled neutral amino acid transporter 2 protein encoded by the SLC38A2 gene. The mRNA and amino acid sequences of human SNAT2 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_018976 (SEQ ID NO: 11) and NP_061849 (SEQ ID NO: 12), respectively, and can be used in this invention.

[0023] In other embodiments, the present invention includes the SMCT family, GLUT5, CAT1, THTR2 and / or SNAT2, as well as the ASCT family, Asc1, PAT1 and ATB 0,+ The D-serine transport regulator may further include one or more D-serine transporter proteins selected from a group of D-serine transporter proteins consisting of the following (referred to as the "second group of D-serine transporter proteins" as appropriate), which controls the transport of D-serine into and out of cells by D-serine transporter proteins. In this specification, "ASCT family" refers to the ASCT protein family, including ASCT1 and ASCT2.

[0024] ASCT1 is a sodium-dependent alanine, serine, cysteine, and threonine transporter 1 protein encoded by the SLC1A4 gene. The mRNA and amino acid sequences of human ASCT1 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_003038 (SEQ ID NO: 13) and NP_003029 (SEQ ID NO: 14), respectively, and can be used in this invention.

[0025] ASCT2 is a sodium-dependent alanine, serine, cysteine, and threonine transporter 2 protein encoded by the SLC1A5 gene. The mRNA and amino acid sequences of human ASCT2 are available, for example, in the GenBank database and the GenPept database as access numbers NM_005628 (SEQ ID NO: 15) and NP_005619 (SEQ ID NO: 16), respectively, and can be used in this invention.

[0026] Asc1 is a sodium-independent alanine, serine, cysteine ​​transporter 1 protein encoded by the SLC7A10 gene. The mRNA and amino acid sequences of human Asc1 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_019849 (SEQ ID NO: 17) and NP_062823 (SEQ ID NO: 18), respectively, and can be used in this invention.

[0027] PAT1 is a proton-coupled amino acid transporter 1 protein encoded by the SLC36A1 gene. The mRNA and amino acid sequences of human PAT1 are available, for example, in the GenBank database and the GenPept database as accession numbers NM_078483 (SEQ ID NO: 19) and NP_510968 (SEQ ID NO: 20), respectively, and can be used in this invention.

[0028] ATB 0,+ This refers to Sodium-andcholoride-dependent neutral and basic amino acid neurporter B, which is encoded by the SLC6A14 gene. 0,+ Human ATB is a protein. 0,+The mRNA and amino acid sequences are provided, for example, in the GenBank database and the GenPept database as acceptance numbers NM_007231 (SEQ ID NO: 21) and NP_009162 (SEQ ID NO: 22), respectively, and can be used in the present invention.

[0029] In one embodiment, the present invention provides a D-serine transport regulator characterized by regulating the amount of D-serine in cells, tissues, organs, or body fluids. In this specification, "regulating the amount of D-serine in cells" means adjusting the amount of D-serine to any range by increasing or decreasing the amount of D-serine in cells by applying the D-serine transport regulator. In this specification, "regulating the amount of D-serine in tissues" means adjusting the amount of D-serine to any range by increasing or decreasing the amount of D-serine in tissues (e.g., renal tubules, glomeruli, etc.) by applying the D-serine transport regulator. In this specification, "regulating the amount of D-serine in organs" means adjusting the amount of D-serine to any range by increasing or decreasing the amount of D-serine in organs (e.g., kidneys, hearts, etc.) by applying the D-serine transport regulator. In this specification, "adjusting the amount of D-serine in body fluids" means adjusting the amount of D-serine within a desired range by increasing or decreasing the amount of D-serine in body fluids (e.g., blood, urine, etc.) by applying a D-serine transport regulator.

[0030] In one embodiment, the present invention provides a D-serine transport regulator characterized by adjusting the amount of D-serine in the blood and / or urine, preferably the D-serine excretion rate calculated from the amounts of D-serine in the blood and / or urine. In this specification, "adjusting the amount of D-serine in the blood" means adjusting the amount of D-serine in the blood to be within a desired range by applying the D-serine transport regulator to increase or decrease it. For example, the amount of D-serine in the blood may be adjusted to be within the range of 0.5 to 3.0 nmol / mL, preferably 0.7 to 2.5 nmol / mL, and more preferably 1.0 to 2.0 nmol / mL. "Adjusting the amount of D-serine in the urine" means adjusting the amount of D-serine in the urine, i.e., the D-serine excretion rate, to be within a desired range by applying the D-serine transport regulator to increase or decrease it. For example, the D-serine excretion rate may be adjusted to be within the range of 20 to 80%, preferably 30 to 70%, and more preferably 40 to 60%.

[0031] In this specification, the "excretion rate of D-serine" is an index indicating the extent to which D-serine is excreted into urine among the amount filtered by the glomerulus, through the regulatory functions of the renal tubules such as reabsorption and secretion, and is represented in any unit other than a ratio or percentage. Also, a value excluding the effects of water reabsorption and concentration can be calculated by correction with a correction factor, and it may be represented as fractional excretion (FE). Since the concentration rate of urine may not be constant, the excretion rate of the target D-serine may be corrected using a "correction factor" for correcting the concentration rate of urine. For example, in one embodiment of the present invention, the excretion rate of D-serine may be corrected with a correction factor derived from blood and / or urine. The excretion rate of D-serine is most simply represented as the ratio of the amount of D-serine in urine divided by the glomerular filtration amount of D-serine, and the glomerular filtration amount obtained from inulin clearance or the like in the calculation, the actually measured urine volume, and the amount of D-serine in blood may be used. For calculating the excretion rate of D-serine, the amount of L-amino acid in urine (preferably, the amount of L-serine) can be used as a urine volume correction factor. Creatinine clearance calculated using the amount of creatinine in urine or the amount of creatinine in blood as a correction factor can be used. For example, the excretion rate of D-serine can be represented by the following formula, and it may be multiplied by 100 and represented as a percentage (%). [Number] [In the formula, U D-Ser represents the amount of D-serine in urine, P D-Ser represents the amount of D-serine in blood, U cre represents the amount of creatinine in urine, P cre represents the amount of creatinine in blood.]

[0032] The amounts of D-serine and L-serine can be measured by any method, for example, by chiral column chromatography, enzymatic methods, or immunological methods using monoclonal antibodies that identify optical isomers of amino acids. The measurement of the amounts of D-serine and L-serine in a sample in this invention may be carried out using any method well known to those skilled in the art. For example, chromatography and enzymatic methods (Y. Nagata et al., Clinical Science, 73 (1987), 105. Analytical Biochemistry, 150 (1985), 238., A. D'Aniello et al., Comparative Biochemistry and Physiology Part B, 66 (1980), 319. Journal of Neurochemistry, 29 (1977), 1053., A. Berneman et al., Journal of Microbial & Biochemical Technology, 2 (2010), 139., WG Gutheil et al., Analytical Biochemistry, 287 (2000), 196., G. Molla et al., Methods in Molecular Biology, 794 (2012), 273., T. Ito et al., Analytical Biochemistry, 371 (2007), 167., etc.), antibody methods (T. Ohgusu et al., Analytical Biochemistry, 357 (2006), 15., etc.), gas chromatography (GC) (H. Hasegawa et al., Journal of Mass Spectrometry, 46 (2011), 502., MC Waldhier et al., Analytical and Bioanalytical Chemistry, 394 (2009), 695., A. Hashimoto, T. Nishikawa et al., FEBS Letters, 296 (1992), 33., H.Biomedical Chromatography, 15 (2001), 166., M. Junge et al., Chirality, 19 (2007), 228., M. Waldhier et al., Journal of Chromatography A, 1218 (2011), 4537. Bruckner and A. Schieber. oxidation) and chemical electrolytes (CE) (H. Miao et al., Analytical Chemistry, 77 (2005), 7190., DL Kirschner et al., Analytical Chemistry, 79 (2007), 736., F. Kitagawa, K. Otsuka, Journal of Chromatography B, 879(2011), 3078., G. Thorsen and J. Bergquist, Journal of Chromatography B, 745(2000),389. oxidation) and high-performance high-performance chromatography (HPLC) (N. Nimura and T. Kinoshita, Journal of Chromatography, 352 (1986), 169., A. Hashimoto et al. [ PMC free article ] [ PubMed ] 41., H. Bruckner et al., Journal of Chromatography A, 666 (1994), 259., N. Nimura et al., Analytical Biochemistry, 315(2003), 262., C. Muller et al., Journal of Chromatography A, 1324 (2014), 109.,S. Einarsson et al., Analytical Chemistry, 59 (1987), 1191., E. Okuma and H. Abe, Journal of Chromatography B, 660 (1994), 243., Y. Gogami et al., Journal of Chromatography B, 879 (2011), 3259., Y. Nagata et al., Journal of Chromatography, 575 (1992), 147., S. A. Fuchs et al., Clinical Chemistry, 54 (2008), 1443., D. Gordes et al., Amino Acids, 40 (2011), 553., D. Jin et al., Analytical Biochemistry, 269 (1999), 124., J. Z. Min et al., Journal of Chromatography B, 879 (2011), 3220., T. Sakamoto et al., Analytical and Bioanalytical Chemistry, 408 (2016), 517., W. F. Visser et al., Journal of Chromatography A, 1218 (2011), 7130., Y. Xing et al., Analytical and Bioanalytical Chemistry, 408 (2016), 141., K. Imai et al., Biomedical Chromatography, 9 (1995), 106., T. Fukushima et al., Biomedical Chromatography, 9 (1995), 10., R. J. Reischl et al., Journal of Chromatography A, 1218 (2011), 8379., R. J. Reischl and W. Lindner, Journal of Chromatography A, 1269 (2012), 262., S. Karakawa et al., Journal of Pharmaceutical and Biomedical Analysis, 115 (2015), 123., etc.).

[0033] The optical isomer separation and analysis system in the present invention may combine multiple separation and analysis methods. More specifically, by using an optical isomer analysis method characterized by including the steps of: separating the components of a sample by passing a sample containing components having optical isomers through a first column packing material as a stationary phase together with a first liquid as a mobile phase; holding each of the components of the sample individually in a multiloop unit; supplying each of the components of the sample held individually in the multiloop unit together with a second liquid as a mobile phase through a flow path to a second column packing material having an optically active center as a stationary phase, thereby separating the optical isomers contained in each of the components of the sample; and detecting the optical isomers contained in each of the components of the sample, the amount of D- / L-amino acids in a sample can be measured (Patent No. 4291628). In HPLC analysis, D- and L-amino acids may be derivatized beforehand using fluorescent reagents such as o-phthalaldehyde (OPA) or 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), or diastereomerized using N-tert-butyloxycarbonyl-L-cysteine ​​(Boc-L-Cys), etc. (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, Vol. 53, 677-690 (2004)). Alternatively, D-amino acids can be measured by immunological methods using monoclonal antibodies that specifically bind to optical isomers of amino acids, such as D-serine and L-serine. Furthermore, when the total amount of D- and L-isomers is used as an indicator, it is not necessary to separate and analyze the D- and L-isomers; amino acids can be analyzed without distinguishing between D- and L-isomers. In this case, separation and quantification can also be performed using enzymatic methods, antibody methods, GC, CE, HPLC, etc.

[0034] In one embodiment, the D-serine transport regulator of the present invention acts on D-serine transporter proteins expressed in kidney cells. This makes it possible to regulate the amount of D-serine in the blood and / or urine, preferably the excretion rate of D-serine.

[0035] In one embodiment, the D-serine transport regulator of the present invention is characterized by inhibiting the transport of D-serine to cells by acting on D-serine transporter proteins. For example, it may be a substrate or inhibitor of D-serine transporter proteins (e.g., a selective inhibitor or a non-selective inhibitor), a substance that inhibits the expression of D-serine transporter proteins themselves, or a substance that directly or indirectly suppresses genes related to the expression of D-serine transporter proteins. In this specification, "selective inhibitor of D-serine transporter proteins" refers to an inhibitor that selectively acts on D-serine transporter proteins and exhibits inhibitory activity. Examples include inhibitors whose Ki value for D-serine transporter proteins is 1 / 5 times, preferably 1 / 10 times, more preferably 1 / 25 times, and even more preferably 1 / 100 times or less, the Ki value for other proteins. The Ki value of a selective inhibitor of D-serine transporter proteins can be measured using various methods well known in the art. Selective inhibitors of D-serine transporter proteins may be, for example, small molecule compounds, aptamers, antibodies, antibody fragments, and combinations thereof. Furthermore, in this specification, "non-selective inhibitor of D-serine transporter protein" refers to an inhibitor that acts non-selectively on D-serine transporter protein and exhibits inhibitory activity.

[0036] In this specification, "low molecular weight compound" means a molecule of a size comparable to organic molecules commonly used in pharmaceuticals, and for example, a compound having a molecular weight in the range of about 5000 Da or less, preferably about 2000 Da or less, and more preferably about 1000 Da or less. In one embodiment, the low molecular weight compound as a D-serine transport regulator of the present invention may be a low molecular weight compound selected from one or more substrates or inhibitors for D-serine transporter proteins selected from the group of first D-serine transporter proteins consisting of the SMCT family, GLUT5, CAT1, THTR2, and SNAT2 (as may be referred to as the "group of first substrates or inhibitors"). In one embodiment, the low molecular weight compound used as the D-serine transport regulator of the present invention may be a substrate or inhibitor of SMCT1, for example, ibuprofen, fenoprofen, ketoprofen, probenecid, acetylsalicylic acid, naproxen, pyroglutamic acid, phenoxyacetic acid, acetic acid, propionic acid, butyric acid, L-lactic acid, D-lactic acid, pyruvate, nicotinic acid, acetoacetic acid, β-D-hydroxybutyric acid, β-L-hydroxybutyric acid, γ-hydroxy The following may be selected, but are not limited, from the group consisting of butyric acid, α-ketoisocaproic acid, benzoic acid, salicylic acid, 5-aminosalicylic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPA), 2-chlorophenoxyacetic acid (2-CPA), 2,3-dichlorophenoxyacetic acid, 3,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid and its derivatives, and pharmaceutically acceptable salts thereof.

[0037] In this specification, “derivative” is intended to include substances in which a part of a specific compound or protein molecule has been modified with various substituents or sugar chains. In this specification, “pharmaceutically acceptable salt thereof” refers to any non-toxic salt formed from a substance used as a D-serine transport regulator. Such salts can be obtained, for example, by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid; organic acids such as oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic acid, and benzylsulfonic acid; inorganic bases such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and ammonium hydroxide; organic bases such as methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, guanidine, choline, and syncholine; or amino acids such as lysine, arginine, and alanine. Furthermore, "medically acceptable salts thereof" include hydrated products and solvates (e.g., hydrates, etc.) of substances used as D-serine transport regulators.

[0038] In one embodiment, the low molecular weight compound used as a D-serine transport regulator of the present invention may be a substrate or inhibitor of SMCT2, and may be selected from, for example, ibuprofen, fenoprofen, ketoprofen, probenecid, acetylsalicylic acid, naproxen, pyroglutamic acid, phenoxyacetic acid and its derivatives, and pharmaceutically acceptable salts thereof, and is not limited thereto.

[0039] In one embodiment, the low molecular weight compound used as a D-serine transport regulator of the present invention may be a substrate or inhibitor of GLUT5, and may be selected from, for example, the group consisting of N-(4-methanesulfonyl-2-nitrophenyl)-2H-1,3-benzodioxol-5-amine (MSNBA), fructose and its derivatives, and pharmaceutically acceptable salts thereof.

[0040] In one embodiment, the low molecular weight compound used as a D-serine transport regulator of the present invention may be a substrate or inhibitor for CAT1, and may be selected from, for example, the group consisting of N-ethylmaleimide (NEM), N-amino-L-arginine, N-amino-L-homoarginine, L-arginine, L-histidine, L-lysine, L-ornithine and its derivatives, and pharmaceutically acceptable salts thereof.

[0041] In one embodiment, the low molecular weight compound used as a D-serine transport regulator of the present invention may be a substrate or inhibitor of THTR2, and may be selected from the group consisting of, for example, metformin, chloroquine, 2,4-diaminopyrimidine, drugs containing a 2,4-diaminopyrimidine group (e.g., fedratinib, AZD1480, cerdulatinib), thiamine and its derivatives, and pharmaceutically acceptable salts thereof.

[0042] In one embodiment, the low molecular weight compound used as a D-serine transport regulator of the present invention may be a substrate or inhibitor of SNAT2, and may be selected from, for example, the group consisting of methyl-amino-isobutyric acid (MeAIB), γ-glutamyl-p-nitroanilide (GPNA), 2-amino-4-bis(alyroxybenzyl)aminobutanoic acid (AABA), L-alanine, L-methionine, L-proline, L-serine, L-asparagine, L-glutamine, L-histidine, glycine and its derivatives, and pharmaceutically acceptable salts thereof.

[0043] In other embodiments, the D-serine transport regulator of the present invention includes one or more small molecule compounds selected from the group of first substrates or inhibitors, in addition to the ASCT family, Asc1, PAT1, and ATB. 0,+The present invention may further include one or more small molecule compounds selected from a group of substrates or inhibitors for D-serine transporter proteins selected from a second group of D-serine transporter proteins consisting of the above (referred to as the "second group of substrates or inhibitors" as appropriate). For example, the present invention may include substances selected from the group consisting of phenylglycine analogs (e.g., L-4-fluorophenylglycine, L-4-chlorophenylglycine, etc.) that are substrates or inhibitors for the ASCT family (e.g., ASCT1 and ASCT2), benzylserine, benzylcysteine, S-benzyl-L-cystine, L-γ-glutamyl-p-nitroanilide, L-serine, L-threonine, L-methionine, L-alanine, L-cysteine, L-glutamine, D-alanine and its derivatives, and pharmaceutically acceptable salts thereof. Furthermore, for example, it may contain a substance selected from the group consisting of phenylglycine analogs (e.g., L-4-bromophenylglycine, L-4-hydroxyphenylglycine, etc.), alanine analogs (e.g., 2-aminoisobutyric acid (AIB), etc.), L-serine, L-alanine, L-cysteine, glycine, L-threonine and its derivatives, and pharmaceutically acceptable salts thereof, which are substrates or inhibitors of Asc1. Furthermore, for example, it may contain a substance selected from the group consisting of taurine, GABA, tryptophan, tryptamine derivatives, 5-hydroxy-L-tryptophan, serotonin, indole-3-propionic acid and its derivatives, and pharmaceutically acceptable salts thereof, which are substrates or inhibitors of PAT1. Furthermore, for example, ATB 0,+ The substance may also include a substance selected from the group consisting of α-methyl-DL-tryptophan, its derivatives, and pharmaceutically acceptable salts thereof, which are substrates or inhibitors of the substance.

[0044] In this specification, "aptamer" refers to synthetic DNA or RNA molecules and peptide molecules that have the ability to specifically bind to a target substance, and can be chemically synthesized in vitro in a short time. The aptamers used in the present invention can, for example, bind to D-serine transporter proteins and inhibit the activity of D-serine transporter proteins. The aptamers used in the present invention can be obtained, for example, by repeatedly selecting the binding to various molecular targets such as small molecules, proteins, and nucleic acids in vitro using the SELEX method (see Turek C., Gold L., Science, 1990, 249(4968), 505-510; Ellington AD, Szostak JW., Nature, 1990, 346(6287):818-822; U.S. Patent No. 6,867,289; U.S. Patent No. 5,567,588; U.S. Patent No. 6,699,843).

[0045] In this specification, "antibody fragment" refers to a portion of a full-length antibody that maintains the activity to bind to an antigen, and generally includes its antigen-binding domain or variable domain. Examples of antibody fragments include F(ab')2, Fab', Fab, or Fv antibody fragments (including scFv antibody fragments). Fragments obtained by treating an antibody with a protease enzyme and, in some cases, by reduction are also included in antibody fragments. The antibody or antibody fragment used in the present invention may be any of the following antibodies: human-derived antibody, mouse-derived antibody, rat-derived antibody, rabbit-derived antibody, camelid-derived antibody such as a llama, or goat-derived antibody. Furthermore, these may be polyclonal or monoclonal antibodies, complete or shortened (e.g., F(ab')2, Fab', Fab, or Fv fragment) antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0046] In one embodiment, the D-serine transport regulator of the present invention may directly or indirectly inhibit the expression of D-serine transporter proteins and may be selected from, for example, small molecule compounds, aptamers, antibodies, antibody fragments, and antisense RNA or DNA molecules, RNAi-inducible nucleic acids, microRNAs (miRNAs), ribozymes, genome-edited nucleic acids, and their expression vectors.

[0047] In this specification, "antisense RNA or DNA molecule" refers to a molecule that has a base sequence complementary to a specific functional RNA (sense RNA), such as messenger RNA (mRNA), and has the function of inhibiting the synthesis of the protein that the sense RNA should carry out by forming a double helix with the sense RNA. In the present invention, an antisense oligonucleotide containing an antisense RNA or DNA molecule inhibits the translation of D-serine transporter protein into protein by binding to the mRNA of the D-serine transporter protein. This reduces the expression level of the D-serine transporter protein and inhibits its activity. Methods for synthesizing antisense RNA or DNA molecules are well known in the art and can be used in the present invention.

[0048] In this specification, "RNAi-inducible nucleic acid" refers to a polynucleotide that can induce RNA interference (RNAi) when introduced into a cell, and is typically RNA, DNA, or an RNA-DNA chimeric molecule containing 19 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 19 to 23 nucleotides, and is optionally modified. RNAi may occur in relation to mRNA, or it may be RNA immediately after transcription before processing, i.e., RNA with a nucleotide sequence including exons, introns, 3' untranslated regions, and 5' untranslated regions. RNAi methods usable in this invention may induce RNAi by methods such as (1) directly introducing short double-stranded RNA (siRNA) into a cell, (2) incorporating small hairpin RNA (shRNA) into various expression vectors and introducing the vector into the cell, or (3) creating a vector that expresses siRNA by inserting short double-stranded DNA corresponding to siRNA between the promoters of a vector having two promoters aligned in opposing directions, and introducing the resulting vector into the cell. RNAi-inducible nucleic acids may include siRNA, shRNA, or miRNA that enable the cleavage or suppression of the function of D-serine transporter proteins. These RNAi nucleic acids may be introduced directly using liposomes or the like, or they may be introduced using expression vectors that induce these RNAi nucleic acids.

[0049] In one embodiment, the RNAi-inducible nucleic acid used in the present invention for D-serine transporter proteins may be any nucleic acid that exhibits a biological effect of inhibiting or significantly suppressing the expression of D-serine transporter proteins, and those skilled in the art can synthesize it by referring to the base sequence of the D-serine transporter protein. For example, it can be chemically synthesized using an automated DNA ( / RNA) synthesizer utilizing DNA synthesis technology such as the solid-phase phosphoamidite method, or it can be synthesized by commissioning an siRNA-related contract synthesis company (e.g., Life Technologies). In one embodiment, the siRNA used in the present invention may be derived from its precursor, short-hairpin double-stranded RNA (shRNA), via processing by the intracellular RNase Dicer.

[0050] In this specification, "microRNA (miRNA)" refers to a single-stranded RNA molecule with a length of 21 to 25 nucleotides that is involved in the post-transcriptional regulation of gene expression in eukaryotes. miRNAs generally recognize the 3'UTR of mRNA, thereby suppressing the translation of target mRNA and inhibiting protein production. Therefore, miRNAs that can directly and / or indirectly reduce the expression level of D-serine transporter proteins are also included in the scope of this invention.

[0051] In this specification, "ribozyme" refers to a general term for enzymatic RNA molecules that can catalyze the specific cleavage of RNA. Some ribozymes are larger than 400 nucleotides, such as the group I intron type and M1 RNA contained in RNase P, but others have an active domain of about 40 nucleotides, known as the hammerhead type or hairpin type (see, for example, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191).

[0052] For example, the self-cleaving domain of hammerhead ribozymes cleaves the 3' side of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is considered important for its activity, and it has been shown that cleavage can also occur at A15 or U15 instead of C15 (see, for example, Koizumi, M. et al., FEBS Lett, 1988, 228, 228). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site, it is possible to obtain a restriction enzyme-like RNA cleavage ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. Those skilled in the art can manufacture such a ribozyme by referring to the following literature: Koizumi, M. et al., FEBS Lett, 1988, 239, 285.; Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.; Koizumi, M. et al., Nucl. Acids Res., 1989, 17, 7059.

[0053] Hairpin-type ribozymes can also be used in this invention. These ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin-type ribozymes (see, for example, Kikuchi, Y. & Sasaki, N., Nucl. Acids. Res., 1991, 19, 6751; Kikuchi, Y., Chemistry and Biology, 1992, 30, 112). By specifically cleaving the transcript of a gene encoding a D-serine transporter protein using a ribozyme, the expression of the D-serine transporter protein can be inhibited.

[0054] In this specification, genome editing nucleic acids refer to nucleic acids used to edit a desired gene in a system utilizing nucleases used for gene targeting. Nucleases used for gene targeting include not only known nucleases but also new nucleases that will be used for gene targeting in the future. For example, known nucleases include CRISPR / Cas9 (Ran, FA, et al., Cell, 2013, 154, 1380-1389), TALEN (Mahfouz, M., et al., PNAS, 2011, 108, 2623-2628), and ZFN (Urnov, F., et al., Nature, 2005, 435, 646-651).

[0055] A CRISPR / Cas9 system using CRISPR / Cas9, which can be used in one embodiment of the present invention, will be described.

[0056] The CRISPR / Cas9 system makes it possible to introduce double-strand breaks at any location in DNA. To use the CRISPR / Cas9 system, at least three elements are required: a protospacer adjacent motif (PAM sequence), guide RNA (gRNA), and Cas proteins (Cas,Cas9).

[0057] A gRNA is designed to form a complementary sequence to a target site adjacent to the PAM sequence (5'-NGG), and then introduced into the desired cells along with a Cas protein. The introduced gRNA and Cas protein form a complex. The gRNA binds to the target sequence on the genome, and the Cas protein, through its nuclease activity, cleaves the double helix of the target genomic DNA.

[0058] Subsequently, cells that have undergone double-strand breaks by nucleases undergo either homologous recombination repair (HDR) or non-homologous end joining (NHEJ). If a suitable DNA fragment (e.g., an HDR repair template) is present in the cell, homologous recombination occurs, allowing for modifications such as deletions, insertions, or disruptions in any desired genome. If an HDR repair template is not present, a few bases may be deleted or added during the NHEJ process. This can cause a frameshift in the protein-coding region, disrupting the protein's reading frame or introducing immature stop codons, ultimately making it possible to knock out the desired protein.

[0059] In one embodiment of the present invention, the genome-edited nucleic acid may be a gRNA that targets a gene encoding a D-serine transporter protein, or a vector expressing the same. In another embodiment, the genome-edited nucleic acid may further include a nucleic acid that expresses a nuclease used for gene targeting. The gRNA and the nuclease used for gene targeting (preferably a Cas protein) may be encoded in the same vector, or they may be encoded in separate vectors. In yet another embodiment, the genome-edited nucleic acid may further include a template nucleic acid for HDR repair.

[0060] In one embodiment, the present invention provides a pharmaceutical composition for treating or preventing diseases associated with elevated levels of D-serine in cells, tissues, organs, or body fluids, comprising a D-serine transport regulator as an active ingredient.

[0061] The D-serine transport regulator of the present invention, or a pharmaceutical composition containing the D-serine transport regulator, can be administered via any route of administration, provided that the concentration at the site of action can be appropriately adjusted. Routes of administration include local administration (on the skin, by inhalation, enema, eye drops, ear drops, nasal, vaginal, etc.), enteral administration (oral, tube, intravenous, etc.), and parenteral administration (intravenous, intraarterial, transdermal, intramuscular, etc.).

[0062] In this specification, “diseases associated with elevated levels of D-serine in cells, tissues, organs, or body fluids” means diseases that correlate with elevated levels of D-serine in cells, tissues, organs, or body fluids, such as kidney disease. In one embodiment, “kidney disease” to which the present invention may be applied includes, for example, conditions involving damage to the glomeruli and / or tubules, such as acute kidney injury, chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis, or polycystic kidney disease, or kidney diseases resulting from systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease, or minimal change nephrotic syndrome.

[0063] In one embodiment, the present invention provides a method for treating or preventing diseases associated with elevated levels of D-serine in cells, tissues, organs, or body fluids, comprising administering a D-serine transport regulator to a subject in need thereof. In this specification, “treatment” means alleviating or eliminating the disease or ailment and / or associated symptoms, which can be evaluated, for example, by confirming the restoration and / or maintenance of glomerular filtration rate. In this specification, “prevention” means preventing the onset of a disease or ailment.

[0064] In one embodiment, the present invention provides a D-serine transport regulator characterized by promoting the transport of D-serine into cells by acting on D-serine transporter proteins.

[0065] In one embodiment of the present invention, a D-serine transport regulator, characterized by promoting the transport of D-serine to cells by acting on a D-serine transporter protein, may be, for example, a vector expressing a D-serine transporter protein, a derivative thereof, or a part thereof, or it may be something that directly or indirectly increases the expression of a D-serine transporter protein, and may be selected from, for example, small molecule compounds, aptamers, antibodies, antibody fragments, and antisense RNA or DNA molecules, RNAi-inducible nucleic acids, microRNA (miRNA), ribozymes, genome-edited nucleic acids, and expression vectors thereof.

[0066] In this specification, "vector" refers to a nucleic acid molecule (carrier) that can transport an inserted nucleic acid molecule into a target such as a cell, and its type and structure are not particularly limited, as long as it can replicate and express the inserted nucleic acid molecule within a suitable host cell. For example, the vector may be selected from the group consisting of plasmid vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, and viral vectors. In one embodiment of the present invention, a known method can be used to introduce the vector into cells.

[0067] A "vector expressing a D-serine transporter protein, its derivative, or a part thereof" refers to a vector into which nucleic acids encoding a D-serine transporter protein, its derivative, or a part thereof are inserted, and which, when introduced into a suitable host cell, can express a D-serine transporter protein, its derivative, or a part thereof. The "vector expressing a D-serine transporter protein or its derivative" that can be used in the present invention includes vectors capable of expressing a D-serine transporter protein having at least 85%, preferably 90%, more preferably 95%, even more preferably 97%, and most preferably 99% or more homology (preferably identity) with the amino acid sequence of the above-mentioned D-serine transporter protein. Furthermore, the "vector expressing a D-serine transporter protein or its derivative" that can be used in the present invention may be a vector capable of expressing a D-serine transporter protein that has at least 85%, preferably 90%, more preferably 95%, even more preferably 97%, and most preferably 99% homology (preferably identical) to the amino acid sequence of the above-mentioned D-serine transporter protein, and in which the amino acid sequence of the substrate (e.g., D-serine) binding site is conserved.

[0068] In this specification, "homology" of two amino acid sequences refers to the ratio of identical or similar amino acid residues that appear at each corresponding site when the two amino acid sequences are aligned, and "identity" of two amino acid sequences refers to the ratio of identical amino acid residues that appear at each corresponding site when the two amino acid sequences are aligned.

[0069] The "homology" and "identity" of two amino acid sequences can be determined, for example, using the Needleman-Wunsch algorithm with the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.00 or later) (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453).

[0070] Furthermore, similar amino acids include, for example, those belonging to the same group in the classification based on structure, properties, side chain type, etc., as described below. Aromatic amino acids: F, H, W, Y; Aliphatic amino acids: I, L, V; Hydrophobic amino acids: A, C, F, H, I, K, L, M, T, V, W, Y; Charged amino acids: D, E, H, K, R, etc. Positively charged amino acids: H, K, R; Loaded amino acids: D, E; Polar amino acids: C, D, E, H, K, N, Q, R, S, T, W, Y; Small amino acids: A, C, D, G, N, P, S, T, V, etc. Miniature amino acids: A, C, G, S; Amino acids with aliphatic side chains: G, A, V, L, I; Amino acids with aromatic side chains: F, Y, W; Amino acids with sulfur-containing side chains: C, M; Amino acids with aliphatic hydroxyl side chains: S, T; Amino acids with basic side chains: K, R, H; Acidic amino acids and their amide derivatives: D, E, N, Q.

[0071] In one embodiment of the present invention, a D-serine transport regulator characterized by promoting the transport of D-serine to cells by acting on D-serine transporter proteins may be selected from the group consisting of, for example, diclofenac, curcumin, activin A, and SMCT family, GLUT5, CAT1, THTR2, SNAT2, and PDZK1 expression vectors.

[0072] PDZK1 is a scaffold protein called PDZ domain containing 1, and it is known that increased expression of this protein increases the expression and activity of SMCT family proteins in the membrane (see Liu Y., et al., Drug Metab Pharmacokinet. 2013;28(2):153-8.; Srivastava S., et al., J Physiol Sci. 2019 Mar;69(2):399-408.). The mRNA and amino acid sequences of human PDZK1 are provided, for example, in the GenBank database and the GenPept database as accession numbers NM_002614 (SEQ ID NO: 23) and NP_002605 (SEQ ID NO: 24). In the present invention, a "vector expressing PDZK1, its derivatives, or a part thereof" refers to a vector into which nucleic acids encoding the PDZK1 protein, its derivatives, or a part thereof are inserted, and which, when introduced into a suitable host cell, can express a D-serine transporter protein, its derivatives, or a part thereof. The "vector expressing PDZK1, its derivatives, or a part thereof" that can be used in the present invention includes vectors capable of expressing a PDZK1 protein having at least 85%, preferably 90%, more preferably 95%, even more preferably 97%, and most preferably 99% or more homology (preferably identity) with the amino acid sequence of the PDZK1 protein described above.

[0073] In one embodiment, the present invention provides a pharmaceutical composition for treating or preventing diseases associated with a decrease in the amount of D-serine in cells, tissues, organs, or body fluids, comprising a D-serine transport regulator as an active ingredient.

[0074] In this specification, “diseases associated with a decrease in intracellular, intratissue, intraorgan, or intrafluid levels of D-serine” means diseases that correlate with a decrease in intracellular, intratissue, intraorgan, or intrafluid levels of D-serine, such as kidney disease. In one embodiment, “kidney disease” to which the present invention may be applied includes, for example, conditions involving damage to the glomeruli and / or tubules, such as acute kidney injury, chronic kidney disease, myeloma kidney, diabetic nephropathy, IgA nephropathy, interstitial nephritis or polycystic kidney disease, or kidney disease resulting from systemic lupus erythematosus, primary aldosteronism, benign prostatic hyperplasia, Fabry disease or minimal change nephrotic syndrome.

[0075] In one embodiment, the present invention provides a method for treating or preventing diseases associated with decreased levels of D-serine in cells, tissues, organs, or body fluids, comprising administering a D-serine transport regulator to a subject in need thereof.

[0076] In one embodiment, the present invention is a method for screening substances that control the transport of D-serine into and out of cells by D-serine transporter proteins, A process in which a candidate substance and D-serine are applied to cells expressing D-serine transporter proteins, and the degree of D-serine transport into and out of the cell is evaluated using the expression of cytotoxicity as an indicator. This provides a method that includes [something].

[0077] In this specification, "candidate substance" refers to a substance to be screened, and may, but is not limited to, small molecule compounds, peptides, proteins, tissue extracts or cell culture supernatants of mammals (e.g., mice, rats, pigs, cattle, sheep, monkeys, humans, etc.), plant-derived compounds or extracts (e.g., herbal extracts, compounds derived from herbal medicines), and microorganism-derived compounds, extracts or culture products.

[0078] In this specification, "manifestation of cytotoxicity" refers to the occurrence of an event that causes some kind of damage to living cells (such as cell death, some kind of alteration or reduction in various functions performed by cells, such as proliferation ability and metabolic capacity). In one embodiment, the cells that may be used in the screening method of the present invention are of animal origin, preferably mammalian origin (for example, human, non-human primates, rodents (mice, rats, hamsters, guinea pigs, etc.), rabbits, dogs, cattle, horses, pigs, cats, goats, sheep, etc.), and more preferably human and non-human primate origin cells. Furthermore, the cells that may be used in the screening method of the present invention may be brain-derived or kidney-derived cells, and are not limited to these.

[0079] In one embodiment, the cells that can be used in the screening method of the present invention may be kidney or brain-derived cells expressing a D-serine transporter protein. In another embodiment, the cells that can be used in the screening method of the present invention may express a D-serine transporter protein selected from a first group of D-serine transporter proteins consisting of the SMCT family, GLUT5, CAT1, THTR2, and SNAT2, or, in addition to the D-serine transporter protein selected from the first group of D-serine transporter proteins, the ASCT family, Asc1, PAT1, and ATB may be expressed. 0,+ One or more D-serine transporter proteins selected from the group of second D-serine transporter proteins consisting of the above may also be expressed.

[0080] In one embodiment, the cells that can be used in the screening method of the present invention may be cells obtained by introducing a vector expressing a D-serine transporter protein from an external source.

[0081] In one embodiment, the screening method of the present invention may be a method for selecting substances that inhibit the transport of D-serine into cells by acting on D-serine transporter proteins, and for screening substances that treat or prevent diseases associated with elevated levels of D-serine in cells, tissues, organs, or body fluids (e.g., kidney disease). Alternatively, in one embodiment, the screening method of the present invention may be a method for selecting substances that promote the transport of D-serine into cells by acting on D-serine transporter proteins, and for screening substances that treat or prevent diseases associated with decreased levels of D-serine in cells, tissues, organs, or body fluids (e.g., kidney disease).

[0082] In one embodiment, the present invention provides a method for screening D-serine transporter proteins using the transport of D-serine into cells as an indicator.

[0083] In one embodiment of the present invention, the "indicator of D-serine transport into cells" can be any indicator that D-serine has been transported into the cell. For example, it may be the amount of radioisotope measured by a scintillation counter or the like when D-serine labeled with a radioisotope is added to cells, or the degree of cellular toxicity caused by the addition of D-serine may be used as the indicator of D-serine transport into cells. When the "indicator of D-serine transport into cells" is the degree of cellular toxicity caused by the addition of D-serine, the assay system becomes simpler and is therefore preferable.

[0084] In one embodiment, the cells used in the present invention may be cells expressing a candidate transporter protein, or, for example, cells obtained by introducing a vector expressing a candidate transporter protein. In this specification, "candidate transporter protein" refers to a protein that is known as a membrane transporter protein but not as a D-serine transporter protein. The gene sequences of candidate transporter proteins that can be applied in the screening method of the present invention can be obtained using known gene databases, etc. Therefore, those skilled in the art can create a vector expressing a candidate transporter protein using known gene databases, etc.

[0085] In one embodiment, the screening method of the present invention may further involve adding an ion selected from the group consisting of sodium ions, potassium ions, protons, and chloride ions, in addition to D-serine. [Examples]

[0086] The present invention will be described in more detail below based on examples, but these examples are not intended to limit the present invention in any way.

[0087] (Example 1) 1.Material General chemicals were purchased from Fujifilm Wako Chemicals Co., Ltd. unless otherwise specified. Cell culture media were obtained from Fujifilm Wako Chemicals Co., Ltd., and bacterial culture media from Nacalai Tesque Co., Ltd. Restriction enzymes were obtained from New England BioLabs in Massachusetts, USA. DNA primer synthesis and DNA sequencing were performed by Macrogen in South Korea. The Flp-In TREx293 cell line was obtained from Invitrogen in California, USA, and fetal bovine serum, p3XFLAG-CMV-14 expression vector, and HRP-conjugated anti-FLAG M2 monoclonal antibody (anti-FLAG-HRP) were purchased from Sigma-Aldrich in Missouri, USA. ASCT2-siRNA was obtained from Thermo Fisher Scientific in Massachusetts, USA. D-[ 3 [H]serine (10 Ci / mmol) was purchased from Moravek, California, USA. Anti-SMCT2(H4) monoclonal antibody was obtained from Santa Cruz Biotechnology, Texas, USA. In the animal experiments, 8-week-old male C57BL / 6 J mice (body weight 21-27g) were purchased from SLC Japan, and these mice were housed in groups of no more than 4 mice per cage under a 12-hour light-dark cycle and fed. The animal experiments were conducted in accordance with the guidelines of the Animal Experiment Utilization Committee of Nara Medical University.

[0088] 2. Experimental Procedure 2-1. Preparation of brush border membrane vesicles (BBMVs) in mouse kidneys After extracting the entire mouse kidney by perfusion with PBS buffer pH 7.4, it was immediately flash-frozen in liquid nitrogen and stored at -80°C until use. The frozen kidney was resuspended in a buffer containing 20 mM Tris-HCl pH 7.6, 250 mM Mannitol, 1 mM EDTA, and cOmplete EDTA-free protease inhibitor cocktail (Roche, Switzerland), and pulverized using PHYSCOTRON (Microtech Nichion Co., Ltd.). The sample was then homogenized using a Potter-Elvehjem homogenizer. After centrifugation at 1,000 × g for 5 minutes at 4°C, the supernatant was centrifuged at 3,000 × g for 5 minutes at 4°C. After centrifugation, the supernatant and 1 M MgCl2 were mixed to a final concentration of 11 mM MgCl2, and incubated on ice for 20 minutes. Subsequently, the mixture was centrifuged at 3,000 × g for 15 minutes at 4 °C, and the supernatant containing crude membrane vesicles was ultracentrifuged at 438,000 × g for 30 minutes at 4 °C. The pellet (BBMVs) remaining after ultracentrifugation was suspended in a buffer containing 20 mM Tris-HCl pH 7.6 and 250 mM Mannitol. The BBMVs were quantified using a BCA protein quantification kit (Thermo Fisher SCIENTIFIC). The BBMVs were stored frozen at -80 °C until use.

[0089] 2-2. Measurement of transport activity using mouse BBMVs Before performing transport activity measurements, to introduce potassium into BBMVs, the frozen BBMVs were thawed, ultracentrifuged at 438,000 × g for 30 minutes at 4°C, and the pellet was suspended in a buffer containing 10 mM Tris-HCl pH 7.6, 100 mM KCl, and 100 mM Mannitol to a protein concentration of 5 mg / mL. The BBMV suspension was incubated on ice overnight. Before starting transport activity measurements, valinomycin was added to the BBMV samples to a final concentration of 5 μM and incubated at room temperature for 30 minutes. Transport activity measurements were performed using an uptake buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl- or KCl instead of NaCl, 50 mM Mannitol, D-[3 The reaction was initiated by diluting the BBMV sample (containing 100 μg) fivefold in a solution containing [H]serine. The reaction mixture was kept warm in a 30°C bath for the instructed time, and the reaction was stopped by adding a well-chilled buffer containing 10 mM Tris-HCl pH 7.6 and 200 mM Mannitol. The mixture was filtered through a 0.45 μm nitrocellulose filter (Millipore) and then washed once with the same buffer. The filtered filter was melted with Clear-sol I (Nacalai Tesque), and the radioactivity on the filter was measured using a β-scintillation counter (LSC-8000, HITACHI). The inhibitor used for the inhibition experiment was D-[ 3 [H]Serine uptake buffer was added simultaneously.

[0090] 2-3. Construction of a cell line that stably expresses FlpIn293TR (1) Cloning of pcDNA5-hSLC5A8 (human SLC5A8 / SMCT1) Currently, the following cDNA sequences for hSLC5A8 are available in the NCBI database: NM_145913 (SEQ ID NO: 1), AF536216 (SEQ ID NO: 25), AF536217 (SEQ ID NO: 26), and AK313788 (SEQ ID NO: 27). AF536216 (SEQ ID NO: 25) has been used in functional analysis studies, and NM_145913 (SEQ ID NO: 1) has been identified from several genome / proteome studies. The cDNA for hSLC5A8_AK313788 (SEQ ID NO: 27) was obtained from the National Institute of Technology and Evaluation (NBRC, NITE). Compared to NM_145913 (SEQ ID NO: 1), AK313788 (SEQ ID NO: 27) contains variations V193I, A201T, and M490I. Therefore, the cDNA clone NM_145913 (SEQ ID NO: 1) was constructed from AK313788 (SEQ ID NO: 27).

[0091] First, the cDNA of hSLC5A8_AK313788 (SEQ ID NO: 27), obtained from NBRC, was incorporated into a p3XFLAG-CMV-14 expression vector to obtain hSLC5A8_AK313788 with a 3xFLAG tag added to the C-terminus. The coding sequence of hSLC5A8_AK313788 (SEQ ID NO: 27) is: 5'-ACTAAGCTTATGGACACGCCACGGGGC-3' (HindIII at the 5' end) (SEQ ID NO: 28), 5'-GCCGGATCCCAAACGAGTCCCATTGCTCTTG-3'(BamHI at the 3' end)(Sequence ID 29) The DNA was amplified by polymerase chain reaction (PCR) using the specified primers. The PCR reaction and thermal cycle profile were performed using Q5 High-Fidelity DNA polymerase (New England BioLabs) according to the manufacturer's protocol: 100 ng of vector template, 0.2 mM dNTPs, 0.5 μM of each primer, and Q5 High-Fidelity DNA polymerase were mixed to a total volume of 50 μL for the PCR reaction. After reacting at 98 °C for 30 seconds, the following cycle was repeated 35 times: 98 °C for 10 seconds, 55 °C at 50% ramp for 30 seconds, and 72 °C for 1 minute. The reaction was then further incubated at 72 °C for 2 minutes.

[0092] PCR products were analyzed by 1% agarose gel electrophoresis, yielding a predicted size of 1.9 kbp. The PCR products were purified using the Gel and PCR clean-up kit (Macherey-Nagel, Germany), and the plasmid vector was extracted using the FavorPrep plasmid extraction mini kit (Favorgen) according to the manufacturer's protocol. The purified plasmid vector and PCR products were cleaved with HindIII and BamHI, and the cleaved PCR inserts were ligated into the cleaved vector using T4 DNA ligase (NEB). The vector and PCR inserts were mixed in an 8:1 ratio and reacted at 16 °C for 1 hour. Subsequently, DH5α-competent E. coli cells (BioDynamics Laboratory) were transformed by heat shock. These cells were cultured on LB medium plates containing 100 mg / L ampicillin at 37 °C for 16 hours. Ampicillin-resistant clones were collected and screened by DNA size screening. DNA size screening was performed by mixing E. coli colonies in lysis buffer (10% w / v sucrose, 100 mM NaOH, 100 mM KCl, 5 mM EDTA, 0.25% w / v SDS, 0.05% w / v bromophenol blue), incubating at 37 °C for 5 minutes, and then analyzing by 0.8% agarose gel electrophoresis. This screening method showed that positive clones had a DNA size larger than the vector backbone on the agarose gel. Finally, the DNA sequence of the positive clones was confirmed. The construct was named "pCMV14-hSLC5A8_AK313788-3xFLAG".

[0093] The next step is to generate "pcDNA5-hSLC5A8_NM145913-3xFLAG" using pcDNA5 / FRT / TO (Invitrogen) as the vector backbone and pCMV14-hSLC5A8_AK313788-3xFLAG as the template, and then generate the insertion fragment of hSLC5A8_NM145913-3xFLAG. Since hSLC5A8_AK313788 has three mutations (V193I, A201T, M490I), the goal was to further mutate these and simultaneously perform subcloning using HiFi DNA Assembly (NEB). Before generating pcDNA5-hSLC5A8_NM145913-3xFLAG, "pcDNA5-hSLC5A8_M490I-3xFLAG" (a clone in which V193 and A201 are mutated to I193 and T201, respectively) was constructed. PCR products were prepared using the following three primer sets.

[0094] 1) 5'-TAAGCTTGGTACCGAGCTCGGCGCGCCATGGACACGCCACGGGGC-3' (Sequence ID 30) 5'-AAATCCAGCCACCATGATCCCAACTTGAAAAACATCTGTCCAG-3' (Sequence ID 31) 2) 5'-TTGGGATCATGGTGGCTGGATTTGCATCCGTGATTATACAGGC-3' (Sequence No. 32) 5'-CCACCAACCATACCAAATACGCTGAGTGCTGCCTGC-3' (Sequence ID 33) 3) 5'-GCGTATTTGGTATGGTTGGTGGACCACTTA-3' (Sequence ID 34) 5'-TTTAAACGGGCCCTCTAGACTCGAGCTACTTGTCATCGTCATCCTTG-3' (Sequence ID 35)

[0095] The PCR reaction was performed using Q5 High-Fidelity DNA polymerase, following the manufacturer's protocol with the following modifications: 100 ng of vector template, 0.2 mM dNTPs, 0.5 μM primers, and Q5 High-Fidelity DNA polymerase were mixed to a total volume of 50 μL. After reacting at 98 °C for 30 seconds, the reaction was repeated 35 times with a cycle of 98 °C for 10 seconds, 55 °C at 50% ramp for 30 seconds, and 72 °C for 30 seconds. The reaction was then continued at 72 °C for 2 minutes. The PCR product was analyzed by 1% agarose gel electrophoresis, yielding a predicted size of 1.9 kbp. After generating three PCR products, linearized pcDNA5 / FRT / TO, to which BamHI + XhoI was added using the HiFi DNA Assembly Kit (NEB), was combined with the vector:insertion in a ratio of 8:1. The mixture was incubated at 50 °C for 1 hour and transformed into DH5α E. coli competent cells by heat shock. Positive clones grown in ampicillin-resistant LB medium were screened by DNA size screening and their sequences were confirmed. Ultimately, "pcDNA5-hSLC5A8_M490I-3xFLAG" (with a 3xFLAG tag added to the C-terminus) was obtained.

[0096] The pcDNA5-hSLC5A8_M490I-3xFLAG was used as a template to induce a mutation from M490 to I490. Site-specific mutation induction was performed using the following primers during PCR.

[0097] 5'-CTACAATGAGACAAATTTGATGACAACCACAGAAATGC-3' (Sequence ID 36) 5'-GCATTTCTGTGGTTGTCATCAAATTTGTCTCATTGTAG-3' (Sequence ID 37)

[0098] PCR reactions were performed using Q5 High-Fidelity DNA polymerase, following the manufacturer's protocol with the following modifications: 100 ng of vector template, 0.2 mM dNTPs, 0.5 μM primers, and Q5 High-Fidelity DNA polymerase were mixed to a total volume of 50 μL. After reacting at 98 °C for 30 seconds, 18 cycles of 98 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 7 minutes were performed. Further reaction was then carried out at 72 °C for 7 minutes. DpnI was added to the PCR product of the entire plasmid, and after incubation at 37 °C for 2 hours, purification was performed using a Gel and PCR clean-up kit. The purified plasmid was transformed into DH5α-competent E. coli cells by heat shock. Subsequently, positive clones were grown in ampicillin-resistant LB medium and their sequences were confirmed. Ultimately, we were able to obtain a positive clone of pcDNA5-hSLC5A8_NM145913-3xFLAG, which we named "pcDNA5-hSLC5A8-3xFLAG".

[0099] (2) Cloning of pcDNA5-hSLC5A12 (human SLC5A12 / SMCT2) hSLC5A12 was amplified from a human kidney cDNA library. This protein was designed to tag the C-terminus with 3xFLAG by cloning the hSLC5A12 cDNA into a p3xFLAG-CMV-14 expression vector. The coding sequence of hSLC5A12 was amplified by PCR using the following primers.

[0100] 5'-TTAGGTACCCATGGAGGTGAAGAACTTTGCAG-3'(KpnI at the 5' end) (Sequence ID 38) 5'-CCGGGATCCGTAGAAATGGGTAGTCTC-3'(BamHI at the 3' end) (Sequence ID 39)

[0101] PCR reactions and thermal cycle profiles were performed using Q5 High-Fidelity DNA polymerase (New England BioLabs), following the manufacturer's protocol with modifications: 50 μL of 100 ng of vector template, 0.2 mM dNTPs, 0.5 μM primers, and Q5 High-Fidelity DNA polymerase were mixed. The mixture was incubated at 98 °C for 30 seconds, followed by 35 cycles of 98 °C for 10 seconds, 55 °C at 50% ramp for 30 seconds, and 72 °C for 1 minute. The mixture was then incubated at 72 °C for 2 minutes. The PCR product was analyzed by 1% agarose gel electrophoresis, yielding a predicted size of 1.9 kbp. The PCR product was purified using the Gel and PCR clean-up kit (Macherey-Nagel, Germany), and the plasmid vector was extracted using the FavorPrep plasmid extraction mini kit (Favorgen) according to the manufacturer's protocol. The purified plasmid and PCR product were cleaved with KpnI and BamHI, and the cleaved PCR inserts were ligated into a linear vector using T4 DNA ligase (NEB). The vector and PCR inserts were mixed in an 8:1 ratio and reacted at 16 °C for 1 hour. Subsequently, DH5α-competent E. coli cells (BioDynamics Laboratory) were transformed by heat shock. These cells were cultured on LB medium plates containing 100 mg / L ampicillin at 37 °C for 16 hours. Ampicillin-resistant clones were collected and screened by DNA size screening in the same manner as for SLC5A8. The obtained positive clones were identified by DNA sequencing and named "pCMV14-hSLC5A12-3xFLAG".

[0102] The plasmid pCMV14-hSLC5A12-3xFLAG was used as a template and inserted into pCDNA5 / FRT / TO by PCR to generate hSLC5A12-3xFLAG. The primers used were as follows:

[0103] 5'-TAAGCTTGGTACCGAGCTCGGCGCGCCATGGAGGTGAAGAACTTTGC-3' (Sequence ID 40) 5'-TTTAAACGGGCCCTCTAGACTCGAGCTACTTGTCATCGTCATCCTTG-3' (Sequence ID 41)

[0104] The PCR reaction and thermal cycling profile were the same as those used for cloning pCMV14-hSLC5A12-3xFLAG. PCR products ending in XhoI and BamHI were cleaved with the corresponding enzymes. The insert was prepared by combining linearized pcDNA5 / FRT / TO, to which XhoI+BamHI had been added using the HiFi DNA Assembly Kit (NEB), in a vector:insert ratio of 1:4. The mixture was incubated at 50 °C for 1 hour and transformed into DH5α-competent E. coli cells by heat shock. Ampicillin-resistant clones were collected and screened on agarose gels by DNA size screening, similar to SLC5A8. Positive clones were identified by DNA sequencing and named "pcDNA5-hSLC5A12-3xFLAG".

[0105] (3) Construction of a cell line that stably expresses FlpIn293TR Flp-In TREx 293 cell lines were cultured at 37°C under 5% CO2 conditions using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / ml Penicillin G, and 100 μg / ml streptomycin (P / S). Three types of stable cell lines were constructed using the corresponding pcDNA plasmid constructs, as described below.

[0106] 1)pcDNA5 / FRT / TO(empty vector) → “FlpIn293TR-Mock”(Mock) 2)pcDNA5-hSLC5A8-3xFLAG → “FlpIn293TR-hSLC5A8-3xFLAG”(hSLC5A8 / hSMCT1) 3)pcDNA5-hSLC5A12-3xFLAG → “FlpIn293TR-hSLC5A12-3xFLAG”(hSLC5A12 / SMCT2)

[0107] To construct stable cell lines, Flp-In TREx293 cells were co-transfected with the corresponding pcDNA5 plasmid construct and the pOG44 Flp-recombinase expression vector (Invitrogen). Transfected cells were seeded in a 1:20 ratio in DMEM + 10% FBS + P / S + 5 mg / L blasticidin + 150 mg / L hygromycin B and cultured. Cells were subcultured three times in a 1:20 ratio to confirm DNA insertion and gene expression. Cells were regularly maintained in the same medium until use. Expression of hSLC5A8 and hSLC5A12 was confirmed in FlpIn293TR-hSLC5A8-3xFLAG and FlpIn293TR-hSLC5A12-3xFLAG by Western blotting and immunofluorescence staining using anti-FLAG antibody and anti-SLC5A12 antibody, respectively. FlpIn293TR-hSLC5A8-3FLAG and FlpIn293TR-hSLC5A12-3xFLAG were expressed in the cell membrane.

[0108] 2-4. Measurement of transport activity in stable cell lines FlpIn293 TR stable cell lines were each placed in 5x10⁶ poly-D-lysine-coated 24-well plates. 4 cells / well (Mock), 6x10 4 Cells (hSLC5A8 and hSLC5A12 cells) were seeded in each well, and 1 mg / L doxycycline hyclate was added 24 hours later to induce recombinant gene expression. The cells were cultured continuously for 2 days, and transport activity was measured at 80-90% confluence. In the ASCT2 knockdown experiment, 10 pmol / well of ASCT2 siRNA was transfected using Lipofectamine 3000 12 hours after cell seeding.

[0109] Before measuring transport activity, the cells were washed three times with transport activity buffer (PBS + 1 g / L D-glucose) warmed at 37°C, and incubated in 500 μL of buffer for 10 minutes at 37°C. Transport activity was measured using the specified concentration of D-[ 3 The reaction was initiated by adding a transport-active buffer containing [H]serine. The reaction mixture was incubated at 37°C for the indicated time. The reaction proceeded with D-[ 3 The reaction was stopped by removing the [H]serine-containing transport activity buffer, and the cells were washed three times with well-chilled transport activity buffer. The cells were lysed in 500 μL of 0.1 N NaOH and incubated for at least 1 hour. The protein concentration of the cell lysates was measured by BCA protein quantification. The lysates were mixed with 1 mL of Emulsifier Safe (PerkinElmer, MA, USA), and the radioactivity of the lysate was measured using a β-scintillation counter (LSC-8000, HITACHI).

[0110] Na + For measuring transport activity in the presence or absence of transport, HBSS(+Na) is used instead of PBS. + :125 mM NaCl, -Na + The following were used: choline chloride, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM D-glucose, and 25 mM HEPES. In inhibition experiments, non-radioactively labeled compounds were included as incorporation substrates, as shown in the figure.

[0111] 2-5.Statistical analysis All experiments were repeated at least four times. Data are expressed as mean ± SEM and tested using an independent t-test (Student's t-test). Statistical analysis was performed using Prism 8.0. *P<0.05, **P<0.01.

[0112] 3.Results 3-1. D-serine transport in BBMV is mainly Na + They were transported dependently (Figure 1).

[0113] D-[ 10 μM (A) and 50 μM (B) in mouse brush border membrane vesicles (BBMVs) 3 The temporal changes in H]serine uptake activity, Na + In the presence of (Na + ), in the absence (K + The D-serine transport system was distinguished by measuring it using Na. Valinomycin was added to a concentration of 5 μM to generate the membrane potential. High D-serine uptake was associated with Na + Observed under the uptake conditions, D-serine transport is mainly Na + We demonstrated that it is driven by dependent transporters.

[0114] 3-2. D-serine transport in BBMV was mainly inhibited by ASCT2 and SMCT inhibitors (Figure 2).

[0115] In mouse BBMV, D-[ in the presence and absence of 1 mM nicotinic acid or 2 mM L-threonine (L-Thr) - 3 [H]Serine transport (10 μM) activity was measured (measurement time: 30 seconds). + Approximately 30% of the dependent uptake was inhibited by nicotinic acid, suggesting that the SMCT transporter contributes to this action. On the other hand, approximately 70% of the uptake activity was inhibited by L-threonine, suggesting that ASCT2 is involved in this action.

[0116] 3-3. Human SMCT1 and hSMCT2 transported D-serine (Figure 3).

[0117] Stable cell lines of FlpIn293TR-hSLC5A8-3xFLAG(hSMCT1) or FlpIn293TR-hSLC5A12-3xFLAG(hSMCT2) were prepared. (A) Flp-In TREx 293 cells were knocked down with ASCT2 siRNA, and expression was confirmed by Western blotting using an anti-ASCT2 antibody. (B) Two days before the uptake experiment, doxycycline was added to hSMCT1 and SMCT2 cells to induce expression. This expression was confirmed by Western blotting using an anti-FLAG antibody. (C) 100 μM D-[ 3 The temporal changes in [H]serine uptake were measured in ASCT2-endogenous hSMCT1 and SMCT2 stable cell lines. (D) Measurements were taken in hSMCT1 and SMCT2 stable cell lines using ASCT2 knockdown. (E) A graph is shown from (D) showing the difference between the observed values ​​in hSMCT1 and SMCT2 stable cell lines and the observed values ​​in mock cells.

[0118] D-[ 3 Uptake of [H]serine was observed in both cell types, indicating D-serine transport mediated by hSMCT1 and SMCT2. A significant difference was observed compared to the control group.

[0119] 3-4. D-serine transport mediated by the SMCT transporter was inhibited by NSAIDs (Figure 4).

[0120] 20 μM D-[ 3 The uptake of [H]serine was measured for 10 minutes in either the FlpIn293TR-hSLC5A8-3xFLAG(hSMCT1) or FlpIn293TR-hSLC5A12-3xFLAG(hSMCT2) stable cell line in the presence of a nonsteroidal anti-inflammatory drug (NSAID): ibuprofen or acetylsalicylic acid. Ibuprofen and acetylsalicylic acid were used in both cell lines. 3 The inhibition of [H]serine transport suggests that these NSAIDs target the hSMCT1 and hSMCT2 transporters. A significant difference was observed compared to the control group.

[0121] 3-5. D-serine transport activity in BBMV was inhibited by the SMCTs inhibitor ibuprofen (Figure 5).

[0122] In the presence of ibuprofen, an SMCT inhibitor, 50 μM D-[ 3 [H]Serine uptake was measured for 1 minute. D-[ 3 [H]serine transport activity was strongly inhibited with 1 and 3 mM ibuprofen.

[0123] (Example 2) 1.Material The following experiment was conducted using the same materials as in Example 1.

[0124] 2. Experimental Procedure 2-1. Measurement of transport activity in HEK 293 and Flp-In TREx 293 cells HEK293 and Flp-In TREx 293 cells were cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / ml Penicillin G, and 100 μg / ml streptomycin (P / S). 6 × 10⁶ cells were placed in a poly-D-lysine coated 24-well plate. 4 Cells were seeded at a rate of cells / well. After culturing for 3 days in DMEM medium supplemented with 10% FBS and P / S, the cells were used for transport activity measurement. Before transport activity measurement, the cells were washed with transport activity measurement buffer (PBS + 1 g / L D-glucose) preheated at 37 °C, and incubated in 500 μL of buffer for 10 minutes at 37 °C. Transport activity measurement was performed using the specified concentration of D-[ 3 The reaction was initiated by adding a [H]serine-containing transport activity buffer. The reaction mixture was incubated at 37°C for the indicated time. The reaction proceeded with D-[ 3The reaction was stopped by removing the [H]serine-containing transport activity buffer, and the cells were washed three times with well-chilled transport activity buffer. The cells were lysed in 500 μL of 0.1 N NaOH and incubated for at least 1 hour. The protein concentration of the cell lysates was measured by BCA protein quantification. The lysates were mixed with 1 mL of Emulsifier Safe (PerkinElmer, MA, USA), and the radioactivity of the lysate was measured using a β-scintillation counter (LSC-8000, HITACHI). + For measuring transport activity in the presence or absence of transport, HBSS(+Na) is used instead of PBS. + :125 mM NaCl, -Na + Choline chloride, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM D-glucose, and 25 mM HEPES were used. In inhibition experiments, non-radioactively labeled compounds were included as incorporation substrates, as shown in the figure.

[0125] 2-2. Construction of a cell line that stably expresses FlpIn293TR A cell line stably expressing FlpIn293TR was constructed using the procedure described in Example 1, Sections 2-3.

[0126] 2-3. Evaluation of protein expression by Western blotting Flp-In TREx293 and FlpIn293TR stable cell lines were cultured in antibiotic-free DMEM + 10% FBS medium for 10 minutes. 6Cells were seeded in a 6cm cell culture plate. The day after culturing, 10 pmol / well of ASCT2 siRNA was transfected using Lipofectamine 3000. Eight hours after transfection, 1 mg / L of doxycycline hyclate was added to promote increased gene expression. Two days after transfection, the cells were washed twice with well-chilled phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4) at pH 7.4. After collection with a cell scraper, the cells were centrifuged to obtain a pellet. The pellet was rapidly frozen in liquid nitrogen. Frozen cells were lysed in PBS containing 1% w / v Fos-Choline-12 (Avanti Polar Lipids, AL, USA), 1% w / v n-dodecyl-β-D-maltoside (DDM; Dojindo Molecular Technologies, Japan), and cOmplete EDTA-free protease inhibitor cocktail (Roche, Switzerland) for 30 minutes. The lysate was centrifuged at 15,000 × g, and the protein content of the supernatant was measured using the BCA protein quantification kit (Thermo Scientific). 50 μg (protein obtained from the lysate) / well was subjected to 10% SDS-PAGE, transferred to a PVDF membrane (Millipore, MA, USA), and blocked using 5% skim milk / TBS-T (Tris buffer saline (20 mM Tris-HCl, 150 mM NaCl), 0.1% v / v Tween-20) or Blocking One (Nacalai Tesque). Primary antibodies used were anti-FLAG-HRP (1:20,000) and anti-ASCT2 (1:2,500). Secondary antibodies used were HRP conjugation (Jackson ImmunoResearch, PA, USA) or StarBright fluorescently labeled secondary antibody (Bio-Rad Laboratories, CA, USA), diluted in Blocking One at a ratio of 1:2,500.The signal of HRP-binding protein was detected using chemiluminescence detection (Immobilon Forte Wstern HRP substrate, Millipore). Images were obtained using the ChemiDoc Touch Imaging system (Bio-Rad Laboratories).

[0127] 2-4. Cell survival measurement D-serine toxicity testing using a transient expression system was performed using HEK293. Cells were seeded at 10,000 cells / well in 96-well plates and transfected with 0.1 g DNA (Mock: pCMV14, SMCT1: pCMV14-hSLC5A8-3xFLAG, SMCT2: pCMV14-hSLC5A12-3xFLAG). D-serine was applied 24 hours after transfection, and the cells were incubated for a further 2 days. The number of proliferated cells was determined by XTT measurement. The XTT solution was prepared by mixing 1 mg / ml of XTT (2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt: Biotium, CA, USA) with 7.5 μg / ml of Phenazine methosulfate (Nacalai Tesque) and adding it to the cultured cells. The reaction was incubated at 37°C in 5% CO2 for 4 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0128] Toxicity testing using stable cells was performed using the FlpIn293TR stable cell line. Cells were placed in poly-D-lysine-coated 96-well plates, each measuring 6.5 x 10⁶. 3 cells / well (Mock), 8x10 3Cells / well (hSLC5A8 and hSLC5A12 cells) were seeded in antibiotic-free DMEM + dialyzed FBS medium. After 24 hours, the cells were treated with different concentrations of L- or D-serine, and 1 mg / L doxycycline hyclate was added to induce recombinant gene expression. ASCT2 knockdown experiments were performed by transfection with 2 pmol / well of ASCT2 siRNA using Lipofectamine 3000 12 hours after cell seeding. The effect of ibuprofen was added simultaneously with the D-serine treatment. The cells were cultured for 2 days until 80% confluence was reached. The number of proliferated cells was determined by XTT measurement as described above.

[0129] 2-5.Statistical analysis Statistical analysis was performed using the same procedure as in Example 1, Sections 2-5.

[0130] 3.Results 3-1. D-serine inhibited the proliferation of Flp-In TREx293 cells (Figure 6).

[0131] Flp-In TREx293 cells were treated with L- or D-serine for 2 days, and cell proliferation was measured by the XTT assay. The same data are shown in (A) linear curve plot and (B) semi-logarithmic plot. EC of reduced cell proliferation due to D-serine treatment. 50 The value was 18.7 mM. D-serine is transported into cells via transporters, and intracellular D-serine was suggested to inhibit cell proliferation. A significant difference was observed in D-serine concentrations between 10 and 40 mM.

[0132] 3-2. D-serine transport is involved in the Na of cell lines. + It was driven by a dependent transporter (Figure 7).

[0133] 10 μM D-[ 3 The temporal changes in [H]serine uptake activity were measured using (A) HEK293 cells and (B) Flp-In TREx 293 cell lines. In both cell lines, Na +High uptake of D-serine was observed under these uptake conditions.

[0134] 3-3. D-serine transport was relatively inhibited by ASCT2 inhibitors (Figure 8). D-[ 3 [H]serine uptake was measured for 10 minutes in the Flp-In TREx 293 cell line in the presence of several inhibitors: 1) ASCT2 nonspecific inhibitors: Benzyl-Cys (S-benzyl-L-cystine) and GPNA (L-γ-glutamyl-p-nitroanilide), 2) ASCT2 substrates: L-serine, L-threonine, L-methionine, 3) system A / N inhibitor: MeAIB (α-Methylaminoisobutyric acid). Uptake activity was inhibited by the ASCT2 nonspecific inhibitors and substrates, but not by the system A / N inhibitor, suggesting that ASCT2 contributes to D-serine uptake in the Flp-In TREx 293 cell line.

[0135] 3-4. Flp-In TREx 293 cells expressed the ASCT2 transporter, and its KD reduced the toxicity of D-serine (Figure 9). (A) Western blotting demonstrated that the ASCT2 transporter is endogenously expressed in Flp-In TREx 293 cells. ASCT2 knockdown in Flp-In TREx 293 cells was performed using ASCT2-siRNA transfection. ASCT2 expression in the membrane fraction of Flp-In TREx 293 cells was detected by Western blotting. This indicates that endogenous ASCT2 expression is highly suppressed in ASCT2 knockdown cells. (B) Flp-In TREx 293 control cells and ASCT2 knockdown cells were treated with L- or D-serine for 2 days, and cell proliferation was measured by XTT. L-serine did not affect either control cell type. On the other hand, the toxicity of D-serine to cell proliferation was mitigated in ASCT2 knockdown cells. This strongly suggests that ASCT2 contributes to intracellular D-serine uptake.

[0136] Compared to D-serine in control cells, a significant effect of D-serine in ASCT2-siRNA-treated cells was observed at concentrations of 20–25 mM.

[0137] 3-5. Toxicity testing of D-serine using transient expression systems of SMCT1 and SMCT2 (Figure 10).

[0138] D-serine toxicity studies were performed in HEK293 cells transiently transfected with (A) pCMV14-hSLC5A8-3xFLAG (SMCT1) or (B) pCMV14-hSLC5A12-3xFLAG (SMCT2). In SMCT1 and SMCT2-expressing cells, D-serine treatment induced higher cytotoxicity compared to mock cells (Mock).

[0139] 3-6. Construction of hSMCT stable cell lines (Figures 11-1 and 11-2) (A) Vector map of pCDNA5-hSLC5A8-3xFLAG for generating stable cell lines of hSMCT1-3xFLAG (B) Vector map of pCDNA5-hSLC5A12-3xFLAG for generating stable hSMCT2-3xFLAG cell lines (C) hSMCT1-3xFLAG (arrowhead) and hSMCT2-3xFLAG (arrow) were subjected to Western blotting using an anti-FLAG antibody. This showed that they expressed proteins corresponding to both stable cell lines.

[0140] 3-7. SMCT2 enhanced the inhibition of proliferation by D-serine (Figure 12).

[0141] The cell proliferation effect of serine treatment was investigated in FlpIn293TR-Mock (Mock) or FlpIn293TR-hSLC5A12-3xFLAG (SMCT2) stable cell lines. L-serine treatment did not affect cell proliferation in either cell line. D-serine treatment resulted in a greater reduction in cell proliferation in SMCT2 cells (■) than in Mock cells (●). These findings indicate that SMCT2 enhances the proliferation-reducing effect of D-serine, suggesting that SMCT2 contributes to D-serine uptake. *A significant effect of D-serine on SMCT2 compared to mock was observed at concentrations of 15–25 mM. P < 0.05.

[0142] 3-8. Ibuprofen reduced D-serine sensitivity in SMCT2 stable cell lines (Figure 13). The cell proliferation effect of D-serine treatment was investigated in FlpIn293TR-Mock (Mock) and FlpIn293TR-hSLC5A12-3xFLAG (SMCT2) stable cell lines. D-serine treatment in SMCT2 cells (●) resulted in a stronger decrease in proliferation compared to mock cells (○). Addition of 500 μM ibuprofen (■), an SMCT2 inhibitor, to SMCT2 cells reduced the D-serine effect to the same level as in mock cells. These results suggest that ibuprofen inhibits D-serine uptake via SMCT2. D-serine accumulation was reduced in SMCT2 stable-expressing cells, indicating that the toxic effects of D-serine were mild.

[0143] Ibuprofen supplementation compared to SMCT2: A significant effect of ibuprofen on SMCT was observed at concentrations of 17.5–20 mM. No significant difference was observed between SMCT2 + 500 μM ibuprofen and mock cells.

[0144] 3-9. SMCT1 increased D-serine sensitivity, and ibuprofen counteracted this increased D-serine sensitivity (Figure 14).

[0145] The cell proliferation effect of D-serine treatment was investigated in FlpIn293TR-Mock (Mock) and FlpIn293TR-hSLC5A8-3xFLAG (SMCT1) stable cell lines. D-serine treatment in SMCT1 cells (●) resulted in a stronger decrease in proliferation compared to mock cells (○). Addition of 500 μM ibuprofen (■), an SMCT1 inhibitor, to SMCT1 cells reduced the D-serine effect to the same level as in mock cells. These results suggest that ibuprofen inhibits D-serine uptake via SMCT1. D-serine accumulation in SMCT1 cells decreased, and the toxic effects of D-serine were mild.

[0146] A significant effect of ibuprofen on SMCT cells with ibuprofen supplementation compared to SMCT1 was observed at concentrations of 10–20 mM. No significant difference was observed between SMCT1 + 500 μM ibuprofen and mock cells.

[0147] (Example 3) 1. Proteomic analysis of the renal brush border membrane fraction derived from IRI mice. Previous studies have shown that a mouse model with renal ischemia-reperfusion injury (IRI) exhibits an imbalance in L- / D-serine homeostasis in urine and serum. It is thought that low activity of D-amino acid oxidase (DAO), likely related to altered renal D-serine transport, leads to a high accumulation of D-serine in serum. This led to the idea of ​​identifying D-serine transporters on the renal apical membrane based on their expression during IRI. We performed proteomic analysis of the renal brush border membrane fraction from the kidneys of ischemia-reperfusion injury (IRI) mice. For all identified proteins, log2 (Fold Change) (8h / 0h) was plotted against statistical significance (P-value -log10) (Figure 15(A)). The transporter plots are shown in Figure 15(B). The known D-serine transporter ASCT2 showed a log2 value of 0.54 and a -log10 P value of 0.80, which were used as the cutoff value (Figure 15). Transporters expressed in the apical membrane and showing greater expression changes (both increases and decreases) than ASCT2 were selected as candidates. As a result, 19 transporters were selected, of which 18 candidates were SLCs and one was MFS (Table 1).

[0148] [Table 1]

[0149] 2. Screening of D-serine transporters using cytotoxicity tests Based on proteomic results, 19 candidate D-serine transporters were selected. Based on the idea that intracellular D-serine accumulated via the function of D-serine transporters induces cytotoxicity, a cytotoxicity test for screening D-serine transporters was developed. HEK293 cells were transiently transfected with cDNA clones, and treated with 15 mM or 25 mM D-serine, respectively, to observe the initial and quiescent (steady-state) stages of toxic effects. Two days after D-serine treatment, cells were subjected to the XTT cell proliferation assay. The D-serine toxicity effect in each component was compared to that of mock cells. A known D-serine transporter, Asc1, endogenously expressed in the measurement membrane, was used as a positive control. The results showed that cells transfected with SMCT2, CAT1, TAT1, and SNAT2 exhibited increased toxicity from 15 mM D-serine treatment (Figure 16(A)). Treatment with 25 mM D-serine resulted in significant toxicity in cells transfected with GLUT5, SMCT1, SMCT2, CAT1, THTR2, and SNAT2 (Figure 16(B)).

[0150] 3. Identification of SNAT2 as a D-serine transporter To identify positive candidates for D-serine transporters, we aimed to elucidate their transport function in cell models. ASCT2 is expressed in several cell lines and has been shown to create a strong background for D-serine accumulation, hindering the identification of novel D-serine transporters. HAP1 cells (Horizon Discovery), a nearly haploid cell line, are advantageous as a cell model. Because they have a single copy of almost all chromosomes, gene modification is easy, their transcriptome analysis is well-established, and several gene knockout cell lines are available. Therefore, we decided to obtain ASCT2 knockout HAP1 cells and use them as a model for D-serine screening.

[0151] Compared to wild-type cells, ASCT2 knockout HAP1 cells showed reduced D-serine uptake (Figure 17(A)). However, background D-serine transport was still observed in ASCT2 knockout HAP1 cells. Proteomic analysis of HAP1 cells (Table 2) was used to detect the expression of both SNAT1 and SNAT2. SNAT1 and SNAT2 belong to the "System A family" of sodium-dependent small amino acid transporters. We hypothesized that SNAT contributes to D-serine transport in HAP1 cells. To test this hypothesis, we investigated the inhibitory effect of the System A inhibitor MeAIB on D-serine transport. The results showed that MeAIB significantly reduced D-serine transport in ASCT2 knockout HAP1 cells (Figure 17(B)), suggesting that either SNAT1 or SNAT2, or both, are involved in D-serine uptake. The detection of SNAT2 in the kidneys of IRI mice, along with the in vitro analysis of D-serine transport by SNAT, strongly suggests that SNAT2 is one of the D-serine transporters.

[0152] [Table 2]

[0153] 4. Screening of D-serine transporters using ASCT2 knockout HAP1 cells To confirm the positive candidate D-serine transporter derived from cytotoxicity studies, transporter function was investigated in ASCT2 knockout HAP1 cells. Cells stably expressing the candidate transporter were constructed. These cells were transfected with an expression vector containing cDNA and maintained in selective medium. D-serine transport was measured for 10 minutes in the presence of MeAIB, which reduces the background of D-serine transport activity induced by SNAT2. An ASCT2 stable cell line was constructed and used as a positive control. As a result, SMCT1 showed the greatest D-serine uptake. SMCT2 and THTR2 slightly increased D-serine uptake (Figure 18).

[0154] The results of D-serine-induced cytotoxicity and uptake studies provided strong indications that SNAT2 and SMCT1 may be novel D-serine transporters (Table 2). Although the positive results for SMCT2 were not statistically significant, SMCT2 may be another promising candidate. Since SMCT2 is another member of the SMCT family and has been reported as a low-affinity transporter, it is likely that SMCT2 can transport D-serine at low affinity. Therefore, SMCT1 and SMCT2 were selected for transport analysis.

[0155] 5. GABA inhibited D-serine transport mediated by ASCT2.

[0156] ASCT2 has been previously reported to be a D-serine transporter. Previous studies were consistent with the results of our experiment in HEK293 cells. In this experiment, we observed the endogenous expression and D-serine transport function of ASCT2. Furthermore, [ 3[H]D-serine transport was inhibited by the ASCT2 substrates L-serine, L-threonine, and L-methionine, but not by MeAIB (Figure 19(A)). In particular, when we investigated the inhibition of D-serine transport by GABA (γ-aminobutyric acid), we found that GABA inhibited D-serine transport. The inhibitory effect of GABA was further measured in HEK293 cells transfected with ASCT2. GABA inhibited D-serine transport more effectively than L-serine transport. 3 [H]D-serine transport was similarly inhibited (Figure 19(B)). These results strongly suggest that GABA interacts with ASCT2 as an inhibitor.

Claims

1. A D-serine transport regulator characterized by inhibiting the transport of D-serine to cells by acting on D-serine transporter proteins, The D-serine transporter protein comprises at least one or more proteins selected from the SMCT family, The D-serine transport regulator comprises at least one or more selected from antisense RNA or DNA molecules of the SMCT family, RNAi-inducible nucleic acids, genome-edited nucleic acids and their expression vectors, and substrates or inhibitors of the SMCT family, and A D-serine transport regulator wherein the substrate or inhibitor of the SMCT family is one or more selected from ibuprofen, fenoprofen, ketoprofen, probenecid, acetylsalicylic acid, naproxen, pyroglutamic acid, phenoxyacetic acid, acetic acid, propionic acid, butyric acid, L-lactic acid, D-lactic acid, pyruvate, nicotinic acid, acetoacetic acid, β-D-hydroxybutyric acid, β-L-hydroxybutyric acid, γ-hydroxybutyric acid, α-ketoisocaproic acid, benzoic acid, salicylic acid, 5-aminosalicylic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chlorophenoxyacetic acid (4-CPA), 2-chlorophenoxyacetic acid (2-CPA), 2,3-dichlorophenoxyacetic acid, 3,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and their derivatives, and pharmaceutically acceptable salts thereof.

2. The D-serine transporter protein further comprises one or more proteins selected from the ASCT family, The D-serine transport regulator further comprises one or more selected from antisense RNA or DNA molecules of the ASCT family, RNAi-inducible nucleic acids, genome-edited nucleic acids and their expression vectors, and substrates or inhibitors of the ASCT family, and The D-serine transport regulator according to claim 1, wherein the substrate or inhibitor of the ASCT family is one or more selected from phenylglycine analogs, L-4-fluorophenylglycine, L-4-chlorophenylglycine, benzylserine, benzylcysteine, S-benzyl-L-cystine, L-γ-glutamyl-p-nitroanilide, L-serine, L-threonine, L-methionine, L-alanine, L-cysteine, L-glutamine, D-alanine, and derivatives thereof, and pharmaceutically acceptable salts thereof.

3. A D-serine transport regulator according to claim 1 or 2, characterized by regulating the amount of D-serine in cells, tissues, organs, or body fluids.

4. A D-serine transport regulator according to claim 1 or 2, characterized by regulating the amount of D-serine in the blood and / or urine.

5. A pharmaceutical composition for treating or preventing diseases associated with elevated levels of D-serine in cells, tissues, organs, or body fluids, comprising a D-serine transport regulator according to any one of claims 1 to 4 as an active ingredient.

6. A D-serine transport regulator characterized by promoting the transport of D-serine to cells by acting on D-serine transporter proteins, The D-serine transporter protein comprises at least one or more proteins selected from the SMCT family, The D-serine transport regulator is selected from the SMCT family, its derivatives or vectors expressing a part thereof, diclofenac, curcumin, and activin A.

7. A D-serine transport regulator according to claim 6, characterized by regulating the amount of D-serine in cells, tissues, organs, or body fluids.

8. A D-serine transport regulator according to claim 6, characterized by regulating the amount of D-serine in the blood and / or urine.

9. A pharmaceutical composition for treating or preventing diseases associated with a decrease in the amount of D-serine in cells, tissues, organs, or body fluids, comprising a D-serine transport regulator according to any one of claims 6 to 8 as an active ingredient.

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