PC synthesis inhibitor and method for screening the same

Abstract

To provide a novel PC synthesis inhibitor that can inhibit synthesis of PC, and a method for screening the inhibitor.SOLUTION: A method for screening PC synthesis inhibitor includes the steps of: severally mixing a first microorganism in which Eki1, Cki1 and Psd2 genes are deleted and a second microorganism in which the genes are not deleted with a candidate substance to prepare a culture medium containing the microorganisms and candidate substance; severally proliferating the first microorganism and the second microorganism in the culture medium; and comparing proliferating amounts of the first microorganism and the second microorganism, and selecting a candidate substance of a culture medium containing the candidate substance as a PC synthesis inhibitor in the case where the proliferating amount of the first microorganism is lower than the proliferating amount of the second microorganism.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to a PC synthesis inhibitor and a screening method therefor. [Background technology]

[0002] Mitochondria are important organelles that synthesize adenosine triphosphate (ATP), which is the energy for cellular activity. Mitochondria are involved not only in intracellular energy production but also in various cellular functions.

[0003] Mitochondria change their structure through repeated fusion and fission. In recent years, it has become known that abnormalities in the control of mitochondrial structure are involved in various diseases, aging, etc., and the mechanisms of mitochondrial fusion and fission have attracted attention.

[0004] In addition, mitochondria, along with the endoplasmic reticulum, are the major sites of phospholipid synthesis. Among phospholipids, phosphatidylcholine (PC) is the most abundant phospholipid that constitutes the cell membrane of eukaryotic cells and is important for cell proliferation. One of the synthetic pathways for PC is known to be the methylation pathway of phosphatidylethanolamine (PE).

[0005] Like PC, PE is a phospholipid that constitutes cell membranes and is important for cell proliferation. As conceptually shown in Figure 1A, PE is synthesized by decarboxylation from phosphatidylserine (PS). Although mitochondria have decarboxylase enzymes, they do not have the ability to synthesize PS. Therefore, PS is transported from the endoplasmic reticulum (ER) through the mitochondrial outer membrane (MOM) to the mitochondrial inner membrane (MIM), where PE is synthesized from the transported PS. PE produced in mitochondria is transported back to the endoplasmic reticulum (ER) and methylated to synthesize PC.

[0006] The transport mechanisms of these phospholipids are reported in detail in Non-Patent Documents 1 and 2. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kojima, R., Endo, T., and Tamura, Y. A phospholipid transfer function of ER-mitochondria encounter structure revealed in vitro. Sci. Rep. 6, 30777. 10.1038 / srep30777 (2016). [Non-Patent Document 2] Tamura, Y., Kojima, R., and Endo, T. Advanced In Vitro Assay System to Measure Phosphatidylserine and Phosphatidylethanolamine Transport at ER / Mitochondria Interface. In Encyclopedia of Biophysics, pp. 57-67. 10.1007 / 978-1-4939-9136-5_6 (2019). Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a novel PC synthesis inhibitor capable of inhibiting PC synthesis and a screening method for the same. [Means for solving the problem]

[0009] The present inventors have found that the above problems can be solved by the following aspects. Aspect 1 A step of mixing a first microorganism in which the Eki1, Cki1, and Psd2 genes are deleted and a second microorganism in which the genes are not deleted with a candidate substance, respectively, to prepare a medium containing the microorganism and the candidate substance; Growing the first microorganism and the second microorganism in said medium, respectively; and a step of comparing the growth amounts of the first microorganism and the second microorganism, and selecting the candidate substance in the medium containing the candidate substance as a PC synthesis inhibitor when the growth amount of the first microorganism is lower than the growth amount of the second microorganism; A method for screening for a PC synthesis inhibitor, comprising: Aspect 2 A screening method according to aspect 1, wherein the first microorganism and the second microorganism are deficient in Pdr1 and / or Pdr3. Aspect 3 The screening method according to aspect 1, wherein the step of examining the amount of proliferation is carried out by measuring the turbidity of the medium. Aspect 4 2. The screening method according to aspect 1, wherein the first microorganism and the second microorganism are each a yeast. Aspect 5 A PC synthesis inhibitor comprising one or more compounds represented by the following formulas (1) to (4): [ka] [ka] [ka] [ka] (In the formula, X1 to X8 each represent a hydrogen atom, a halogen atom, or a C1 to C6 group which may have a heteroatom. 12 or a chemical group whose terminal group may have a heteroatom selected from the group consisting of a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, and an amino group; n1 to n8 are each an integer from 1 to 5, and Y1 and Y2 each represent a hydrogen atom or a C1-C 12 (Hydrocarbon radical). Aspect 6 6. The PC synthesis inhibitor according to embodiment 5, which is a fungicide or an antibiotic. Aspect 7 6. The PC synthesis inhibitor according to embodiment 5, which is a mitochondrial mitogen. Effect of the Invention

[0010] According to the present invention, it is possible to provide a novel PC synthesis inhibitor capable of inhibiting PC synthesis and a screening method thereof. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1A shows a schematic diagram of the mechanism of PE and PC synthesis in cells. [Figure 1B] Figure 1B shows the results of an experiment in which the reference 2Δ strain, in which only Pdr1 and Pdr3 were deleted, and the 5Δ strain, in which Eki1, Cki1, and Psd2 were additionally deleted in addition to Pdr1 and Pdr3, grew similarly under control conditions. [Figure 1C] FIG. 1C shows the results of screening 3,200 candidate compounds that inhibit the growth of the 5Δ strain. [Figure 1D] FIG. 1D shows the results of comparing the growth results between the 5Δ and 2Δ strains and screening for candidate compounds that inhibit only the growth of the 5Δ strain. [Figure 2A] FIG. 2A shows the chemical structures of four compounds PCiB-1 to 4 obtained by screening. [Figure 2B] FIG. 2B shows the chemical structures of compounds that did not inhibit the growth of the 5Δ strain among the compounds having chemical structures similar to the four compounds PCiB-1 to 4 in FIG. 2A. [Figure 2C] FIG. 2C shows the effect of changing the concentrations of PCiB-1 to 4 on PDME and PC synthesis. [Figure 3A]Figure 3A shows the results of metabolic labeling of phospholipids in the 5Δ strain with 32P-phosphate and analysis by thin-layer chromatography (TLC) in the presence or absence of PCiB compound, showing the content of each type of phospholipid relative to the total amount of phospholipid. [Figure 3B] FIG. 3B shows the results of culturing the wild-type, Cho1Δ, Psd1Δ, Cho2Δ, and Opi3Δ strains in SCD medium containing 32P-phosphate, and extracting total phospholipids from whole cells and separating them by TLC. [Figure 3C] FIG. 3C shows the content of various phospholipids relative to the total amount of phospholipids by the various strains in FIG. 3B. [Figure 4A] FIG. 4A shows the results of visualization of mitochondria of the 5Δ strain cultured with each PCiB compound, observed with a confocal laser microscope. [Figure 4B] FIG. 4B shows the results of observation similar to that of FIG. 4A regarding the presence or absence of Dnm1-109 expression in the Cho2Δ strain. [Figure 4C] FIG. 4C shows the results of quantification of tubular or fragmented mitochondrial morphology. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In one embodiment, a method for screening PC synthesis inhibitors includes the steps of: mixing a first microorganism lacking the Eki1, Cki1, and Psd2 genes, and a second microorganism not lacking the genes, with a candidate substance to prepare a medium containing the microorganism and the candidate substance; growing the first microorganism and the second microorganism in the medium; and comparing the growth rates of the first microorganism and the second microorganism, and selecting the candidate substance of the medium containing the candidate substance as an antibiotic if the growth rate of the first microorganism is lower than the growth rate of the second microorganism.

[0013] As mentioned above, there are multiple pathways by which PC can be synthesized in cells. Figure 1A shows an overview of these pathways.

[0014] The Eki1 and Cki1 genes play an important role in the pathway for the synthesis of PE and PC, known as the Kennedy pathway. The Kennedy pathway begins with the conversion of ethanolamine and choline to phosphatidylethanolamine (P-Etn) and phosphatidylcholine (P-Cho) by Eki1 and Cki1, and then CDP-Etn and CDP-Cho generated from P-Etn and P-Cho react with diacylglycerol present in the endoplasmic reticulum (ER) to generate PE and PC, respectively.

[0015] Among the pathways by which PE and PC are synthesized, this pathway is not the main pathway, and in the screening method of this embodiment, the Eki1 gene and Cki1 gene are deleted from the first microorganism in order to prevent PE and PC from being synthesized via such a pathway.

[0016] In addition, the Psd2 gene plays an important role in synthesizing PE by decarboxylation of PS outside mitochondria.

[0017] Among the pathways by which PE is synthesized, this pathway is also not a major pathway, and in the screening method of this embodiment, the Psd2 gene is deleted from the first microorganism in order to prevent PE from being synthesized through such a pathway.

[0018] The Psd1 gene is involved in the decarboxylation of PS in mitochondria, which is the main pathway of PC synthesis, in which PS from the endoplasmic reticulum is decarboxylated in mitochondria to synthesize PE, and then PE is methylated again in the endoplasmic reticulum to synthesize PC. Three methylations are required for the conversion of PE to PC, but PE methylation in the endoplasmic reticulum occurs in two steps. The first step is methylation of PE by Cho2, which adds one methyl group. The second step is methylation of PE by Opi3, which adds two methyl groups. The Psd1 gene is a very important gene because it generates most of the PE in mitochondria and cells.

[0019] In this embodiment, the first microorganism, which is capable of synthesizing PC by having the Psd1 gene while having the Eki1, Cki1, and Psd2 genes deleted, is mixed with a candidate substance and grown. Similarly, the second microorganism, which is not deleted from these genes, is mixed with a candidate substance and grown. If both the first microorganism and the second microorganism grow in the same way, it can be concluded that the candidate substance does not inhibit the main pathway of PC synthesis. On the other hand, if the second microorganism grows and only the first microorganism does not grow, at least one of the mechanisms of the main pathway of PC synthesis is inhibited in the first microorganism.

[0020] It is further preferred that the first and second microorganisms are deficient in Pdr1 and Pdr3, respectively. These genes are transcription activators encoding drug efflux pumps, and are useful for screening candidate compounds, since they can enhance the effect of the candidate compound.

[0021] These gene deletion methods can be carried out based on the methods described in the Examples and well-known techniques.

[0022] The microorganism is not particularly limited as long as it synthesizes PC intracellularly, and examples thereof include filamentous fungi, yeast, fungi, etc., and yeast can be particularly used. The first microorganism and the second microorganism can be the same except that the first microorganism is deficient in the Eki1, Cki1, and Psd2 genes.

[0023] The medium is not particularly limited as long as it can culture and grow the microorganism used, and various media such as YM medium, YPD medium, YM medium, LB medium, NB medium, SCD medium, etc. The culture conditions are also not particularly limited as long as the second microorganism can grow, and well-known culture conditions can be used.

[0024] The step of comparing the amount of growth is not particularly limited as long as the amount of growth can be evaluated. For example, the step can be performed by optically measuring the turbidity of a transparent container (e.g., a glass container such as a test tube, or a transparent resin container) containing a medium in which a microorganism is grown. For example, OD 600 The amount of growth can be compared by measuring the turbidity. As growth progresses in a culture medium containing microorganisms, the turbidity increases, so the amount of growth can be compared by measuring the turbidity.

[0025] The present inventors have investigated and found that the PC synthesis inhibitors obtained by screening are at least a plurality of compounds, and inhibit the synthesis of PC by inhibiting the methylation of PE by Cho2, particularly, in the main pathway of PC synthesis. Of the PE methylation enzymes Cho2 and Opi3 possessed by microorganisms, Cho2 has been lost in the evolutionary process in higher organisms such as mammals. Therefore, the PC synthesis inhibitors obtained by screening can selectively prevent the growth of microorganisms such as fungi, and can be used as antibacterial agents or antibiotics. Thus, the PC synthesis inhibitors can be said to be inhibitors of PE methylation by Cho2.

[0026] Furthermore, the present inventors have found that PC biosynthesis is important for regulating mitochondrial division, and that PC synthesis inhibitors function as mitochondrial division promoters. The mitochondrial division promoters can be used as research reagents in various situations.

[0027] For example, it has been reported that when mitochondrial division does not proceed normally, the mitochondria themselves become highly bound and are unable to move into narrow spaces such as the neurites of nerve cells, resulting in abnormal synapse formation, etc. By applying a mitochondrial division promoter to cells in which mitochondrial division does not proceed normally, it is possible to examine whether neuronal function is restored.

[0028] In addition, since mitochondria also fuse, this can be used to promote mitochondrial division, for example, by observing mitochondrial fusion after promoting mitochondrial division using a mitochondrial division promoter.

[0029] Furthermore, for example, when analyzing the function of gene A, mitochondrial fragmentation can occur in gene A-deficient cells. In such cases, it is necessary to distinguish whether we are looking at the direct effect of gene A deficiency or the secondary effect of mitochondrial fragmentation. In such cases, the effect of mitochondrial fragmentation using a mitogen can be examined independently of the gene deficiency.

[0030] Specific compounds that can be used as PC synthesis inhibitors include the following four types of compound groups:

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] [ka]

[0035] In the formula, X1 to X8 each represent a hydrogen atom, a halogen atom, or a C1 to C6 group which may have a heteroatom. 12 or a chemical group whose terminal group may have a heteroatom selected from the group consisting of a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, and an amino group.

[0036] C1-C, which may have a heteroatom 12 The hydrocarbon group may be a hydrocarbon group containing a divalent functional group containing a heteroatom, such as an ether group, an ester group, a thioether group, a thioester group, an amide group, a secondary amino group, or a sulfonyl group, and the hydrocarbon portion may be a straight-chain or branched-chain hydrocarbon group, or a cyclic hydrocarbon group such as an aryl group. 12 The hydrocarbon group is a C1-C group containing a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a thiol group, an amino group, etc. at the end. 12 The hydrocarbon group may be a terminal group, and may further include a divalent functional group containing a hetero atom as described above. 12 The hydrocarbon group may be a heterocyclic group.

[0037] The chemical group having an end group selected from the group consisting of a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, a thiol group, and an amino group, which may have a heteroatom, may simply be the end group, or may further have a divalent functional group containing a heteroatom as described above. The chemical group does not have to contain a hydrocarbon group.

[0038] Each of n1 to n8 is an integer of 1 to 5, and may be 1 to 4, 1 to 3, 1 to 2, or 1.

[0039] Y1 and Y2 each represent a C1-C alkyl group which may have a hydrogen atom or a heteroatom. 12 It is a hydrocarbon group of C1 to C which may have a heteroatom. 12 The hydrocarbon group may be the same as those described for X1 to X8.

[0040] The compound of formula (1) corresponds to PCiB-1 discovered by screening in the Examples, where X1 can be a halogen atom, particularly fluorine or chlorine, and n1 can be 1 to 3. Furthermore, X2 can be a C1 to C6 hydrocarbon group (particularly an alkyl group) containing an ether group or a thioether group, and n2 can be 1 to 2.

[0041] Among other mechanisms of inhibition of PC synthesis, the compound of formula (1) can inhibit PE transport from mitochondria to the endoplasmic reticulum, resulting in defective PC synthesis. The compound of formula (1) can also be used as a mitochondrial division promoter.

[0042] The compound of formula (2) corresponds to PCiB-2 discovered by screening in the Examples, where X3 can be an alkylsulfamoyl group (-SO2NR2, where R is an alkyl group of C1 to C3), and n3 can be 1 to 2. X2 can be a halogen atom, particularly chlorine, and n4 can be 1 to 3.

[0043] The compound of formula (3) corresponds to PCiB-3 discovered by screening in the Examples, where X5 can be a halogen atom, particularly fluorine or chlorine, and n5 can be 1 to 3. X6 can be a sulfo group or a nitro group, particularly a nitro group, and n6 can be 1 to 2. Y1 can be hydrogen, C1 to C 12 The substituents may be hydrocarbon radicals, in particular aryl or phenyl radicals.

[0044] The compound of formula (4) corresponds to PCiB-4 discovered by screening in the Examples, and X7 is hydrogen, C1 to C 12 X8 can be a halogen atom, particularly fluorine or chlorine, and n8 can be 1 to 3. Y2 can be hydrogen, a C1 to C3 alkyl group, or a C1 to C3 alkyl group, and n7 can be 1 to 3. ... 12It may be a hydrocarbon group, in particular hydrogen.

[0045] The compounds of formulae (2) to (4) can cause defects in PC synthesis by inhibiting PE methylation by Cho2, among other mechanisms of inhibition of PC synthesis. The compounds of formulae (2) to (4) can also be used as mitochondrial division promoters.

[0046] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. EXAMPLES

[0047] Screening Experiments To screen for small molecules that inhibit phospholipid biosynthesis, we generated yeast mutants in which PE and PC synthesis is highly dependent on transport of PS and PE between the endoplasmic reticulum (ER), the outer mitochondrial membrane (MOM), and the inner mitochondrial membrane (MIM).

[0048] Specifically, we created yeast strains lacking the Eki1 and Cki1 genes, which are important for PE and PC synthesis via the Kennedy pathway, and the Psd2 gene, which codes for the decarboxylation of PS outside of mitochondria.

[0049] Furthermore, the Pdr1 and Pdr3 genes, which are transcriptional activators that encode drug efflux pumps, were deleted from the yeast to enhance the effects of candidate compounds.

[0050] When grown in normal medium without lyso-PE, cells lacking Pdr1, Pdr3, Eki1, Cki1, and Psd2 (referred to as "5Δ strain") produce PE only via Psd1 in the inner mitochondrial membrane (MIM). The 5Δ strain also synthesizes PC only by methylating PE synthesized in mitochondria with the methyltransferases Cho2 and Opi3 present in the endoplasmic reticulum (ER).

[0051] Dilutions of the 5Δ strain and a reference cell line lacking only Pdr1 and Pdr3 (referred to as the “2Δ strain”) were spotted onto YPD plates containing DMSO and cultured at 30°C for 2 days. We confirmed that the 2Δ strain and the 5Δ strain grew similarly under control conditions (Figure 1B).

[0052] Therefore, when the 2Δ and 5Δ strains are mixed and grown with a candidate compound that prevents their growth, a candidate compound that does not inhibit the growth of the 2Δ strain but inhibits the growth of the 5Δ strain can be said to inhibit a major step in the synthesis of PC (i.e., the PE or PC synthesis step via phospholipid transport between the endoplasmic reticulum (ER) and mitochondria).

[0053] To screen for candidate compounds that inhibit this PE or PC synthesis step, 5Δ strain suspensions were dispensed into dozens of assay-ready 384-well plates containing a different candidate compound in each well, grown for 24 h, and then the OD was measured as an index of cell proliferation. 600 was measured.

[0054] Figure 1C shows representative results obtained from testing 3200 candidate compounds (10 plates). The horizontal axis of the graph in Figure 1C is the number of the candidate compound, and the vertical axis shows the results of inhibiting the growth of the 5Δ strain. Among the compounds that strongly inhibited the growth of the 5Δ strain, those that did not inhibit the growth of the 2Δ strain were further screened (Figure 1D).

[0055] In vitro testing A library of over 210,000 small molecule compounds provided by the University of Tokyo Graduate School of Pharmaceutical Sciences, Drug Discovery Institute was used as candidate compounds. Detailed experimental conditions are described in the "Experiment" section at the end of this article.

[0056] As a result, we were able to obtain four compounds that inhibit PC synthesis. We named these compounds PCiB-1 to 4, and their structures are shown in Figure 2A. Among the more than 210,000 small molecule compounds screened, compounds that have similar structures to the four compounds obtained by screening but do not inhibit PC synthesis are shown in Figure 2B.

[0057] In addition, the growth of the 2Δ and 5Δ strains on agar plates by PCiB-1 to 4 was re-examined, and it was confirmed that there was no specific toxicity to the 5Δ strain. PS synthesis was not affected at all in the presence of the PCiB compounds, indicating that Cho1 is not the target of the PCiB compounds. Similarly, it was confirmed that PE was normally produced and even accumulated in the presence of the PCiB compounds. This indicates that Psd1 activity is not affected by the PCiB compounds.

[0058] The effects of varying the concentrations of PCiB-1–4 on phosphatidyldimethylethanolamine (PDME) and PC synthesis were investigated. The calculated IC 50 The values ​​were 7.0 μM, 2.0 μM, 0.4 μM, and 6.8 μM, respectively (FIG. 2C).

[0059] In vivo testing As mentioned above, PCiB-1–4 showed clear inhibition of PC synthesis in vitro. We next investigated whether they could exert the same effect in vivo.

[0060] Phospholipids in the 5Δ strain were 32 The phospholipids were metabolically labeled with P-phosphate and analyzed by thin-layer chromatography (TLC) to investigate the content of each phospholipid relative to the total amount of phospholipids in the presence or absence of PCiB compounds. Here, the 5Δ strain was incubated in the presence of 30 μM PCiB-1 to 4. 32 The cells were cultured in SCD medium containing P-phosphate for 6 hours.

[0061] The results are shown in Figure 3A. In the in vivo study, treatment with PCiB-1 and 2 caused similar effects as in the in vitro study, slightly decreasing and increasing the amounts of PC and PS relative to total phospholipids, respectively. Furthermore, PCiB-2 caused a slight accumulation of PE.

[0062] Treatment with PCiB-3 and 4 showed clear effects in vivo, decreasing the relative amount of PC by approximately 80% and 60%, respectively. Furthermore, when the 5Δ strain was treated with PCiB-3 and 4, rapid accumulation of PE and phosphatidylinositol (PI) was observed. On the other hand, it was confirmed that the PCiB analogs shown in Figure 2B did not show any inhibitory effect on PC accumulation.

[0063] These results indicate that PCiB-1 and 2 were not as effective as PCiB-3 and 4 in vivo, but confirmed that the PCiB compounds are inhibitors of PC synthesis.

[0064] 《Elucidation of the inhibitory mechanism of PC synthesis by PCiB compounds》 Next, to predict the target molecules of PCiB compounds, we analyzed the phospholipid composition of yeast cells lacking enzymes involved in PC biosynthesis. 32 The wild-type, Cho1Δ, Psd1Δ, Cho2Δ, and Opi3Δ strains were metabolically labeled with P-phosphate and the RI-labeled phospholipids were analyzed by TLC. 32 After culturing for 6 h in SCD medium containing P-phosphate, total phospholipids were extracted from the whole cells and separated by TLC, and the content of each type of phospholipid relative to the total amount of these phospholipids was investigated.

[0065] The results are shown in Figure 3B and Figure 3C. As expected, the levels of PS and PE relative to total phospholipids were significantly decreased in Cho1Δ and Psd1Δ strains, respectively, whereas the relative levels of PC were not. Taken together with the in vitro results, Cho1 and Psd1 are unlikely to be targets of the PCiB compounds.

[0066] On the other hand, in the Cho2Δ strain, the relative PC level was greatly decreased, whereas the PE and PI levels were increased, compared with the wild type. The phospholipid profile of the Cho2Δ strain was similar to that of cells treated with PCiB-2 to 4 (Figure 3A and Figure 3C), suggesting that Cho2 is the target of the PCiB compounds. Opi3 was not the target, as phosphatidyl monomethylethanolamine (PMME), a characteristic phospholipid of the Opi3Δ strain, did not accumulate in PCiB-treated cells (Figure 3B).

[0067] In vitro and in vivo studies suggest that PCiB compounds inhibit either the conversion of PE to PC, PE transport from mitochondria to the endoplasmic reticulum (ER), or steps involved in PE methylation.

[0068] We therefore investigated whether the PCiB compounds inhibited the PE methylation process. To directly assess PE methylation, membrane fractions isolated from wild-type cells or Opi3Δ strains were incubated with 100 μg / ml of the methyl donor for PE methylation. 3 Incubated with HS-adenosylmethionine (SAM), 3 The synthesis of 3H-labeled PMME, PDME, and PC was analyzed by TLC.

[0069] When wild-type membrane was used, 3 The amounts of H-labeled PMME, PDME, and PC were significantly decreased in the presence of PCiB-2-4, whereas they remained unchanged in the presence of PCiB-1. These results indicate that PCiB-2-4, but not PCiB-1, 3 These results suggest that it inhibits Cho2 activity that incorporates H-SAM into PE.

[0070] To support this, we used a membrane fraction lacking Opi3, which methylates PMME, in the presence of PCiB-2–4. 3 We observed that the synthesis of H-labeled PMME was significantly inhibited. These results indicate that Cho2 activity is inhibited in the presence of PCiB-2-4.

[0071] On the other hand, PCiB-1 did not have a strong effect on PE methylation. Because PCiB-1 clearly inhibited PC synthesis in vitro (Fig. 2C), it is possible that PCiB-1 slows down PE transport from mitochondria to the endoplasmic reticulum (ER), resulting in a defect in PC synthesis, and indeed the experimental results suggest this.

[0072] <<The effect of PCiB compounds in promoting mitochondrial division>> Since we obtained the PCiB compound that inhibits PC biosynthesis, we decided to use it to analyze the physiological significance of PC. In particular, we analyzed the role of PC in mitochondrial morphogenesis because other phospholipids such as PE, cardiolipin (CL), and phosphatidic acid (PA) have been reported to regulate mitochondrial fusion and fission.

[0073] Specifically, mitochondria of the 5Δ strain were visualized by expressing mitochondrial-targeted GFP (Su9-GFP), and were then cultured with each PCiB compound for 6 hours, after which they were observed under a confocal laser microscope. The results are shown in Figure 4A. The presence or absence of Dnm1-109 expression in the Cho2Δ strain was observed in the same manner as in Figure 4A, and the results are shown in Figure 4B. Figure 4C shows the results of quantifying the morphology of tubular or fragmented mitochondria. At least 100 cells were visualized in each experiment.

[0074] Interestingly, we found that treatment of the 5Δ strain with any of the PCiB compounds resulted in significant fragmentation of mitochondria (Figures 4A and 4C). These results suggest that PC plays a role in mitochondrial morphogenesis. In support of this, we observed that approximately 60% of the Cho2Δ strains exhibited fragmented mitochondria (Figures 4B and 4C).

[0075] Mitochondrial fragmentation is caused by either enhanced mitochondrial fission or impaired mitochondrial fusion. To distinguish between these possibilities, we observed mitochondria in 5Δ or Cho2Δ strains expressing Dnm1-109, a dominant-negative mutant that inhibits Dnm1 function. Interestingly, when Dnm1-109 was expressed, most 5Δ strains treated with PCiB compounds were observed to contain elongated tubular mitochondria.

[0076] Similarly, connected tubular mitochondria were observed in almost all Cho2Δ strains expressing Dnm1-109. These results clearly indicate that mitochondrial fusion occurs normally in Cho2Δ or PCiB-treated 5Δ strains, i.e., PCiB-induced mitochondrial fragmentation is due to the promotion of mitochondrial fission.

[0077] Normal PC biogenesis controls mitochondrial fission. It is known that when Dnm1 is labeled with a fluorescent protein, oligomerized Dnm1 is detected as multiple punctate signals on mitochondria, but Dnm1 oligomers exist in a stationary state, and mitochondrial fission does not occur frequently at these sites. Although what triggers mitochondrial fission is not fully understood, it has been reported that CL on the mitochondrial outer membrane (MOM) activates Dnm1 / Drp1 and promotes mitochondrial fission. On the other hand, Dnm1 / Drp1 bound to PA and saturated fatty acids is inactivated. In this experiment, we showed that PC is another important regulator of Dnm1-dependent mitochondrial fission.

[0078] <<Experiment details>> The Saccharomyces cerevisiae FY833 strain (MATa ura3-52 his3-Δ200 leu2-Δ1 lys2-Δ202 trp1-Δ63) was used as the background strain. The yeast strains used in this experiment are listed in Table 1.

[0079] [Table 1]

[0080] The Pdr1, Pdr3, Cki1, Eki1, Psd2, Opi3, and Cho2 genes can be deleted by homologous recombination using the appropriate gene cassettes amplified from the plasmids listed in Table 2. This resulted in the 2Δ strain, which is deleted for only Pdr1 and Pdr3, and the 5Δ strain, which is deleted for Eki1, Cki1, and Psd2 in addition to Pdr1 and Pdr3.

[0081] [Table 2]

[0082] Primer pairs #NU1080 / 1081, #NU1084 / 1085, #YU1143 / 1144, #NU509 / 510, #NU403 / 404, #YU1637 / 1638, #YU1639 / 164 were used to amplify gene cassettes for deletion of Pdr3, Cki1, Eki1, Psd2, Opi3, and Cho2 genes, the sequences of which are shown in Table 3 below.

[0083] [Table 3]

[0084] Yeast cells were grown in YPD medium (1% yeast extract, 2% polypeptone, and 2% glucose by weight) and SCD medium (0.67% amino acid-free yeast nitrogen base, 0.5% casamino acids, and 2% glucose by weight) containing the appropriate amino acid supplements.

[0085] To obtain yeast growth curves, yeast cultures were prepared as described below: three independent colonies of the 5Δ strain, containing the vector or the 2μ-plasmid, were inoculated into SCD medium and grown for 24 h.

[0086] The culture was grown in SCD medium until the turbidity reached OD 600 = 0.01, and 100 μl aliquots were dispensed into 96-well plates containing candidate compounds. The 96-well plates were incubated at room temperature with shaking on an orbital mixer at 425 cpm and the OD was measured every 20 min for 60 h using a microplate spectrophotometer (Epoch 2, BioTek). 600 was measured.

[0087] In addition, the 2Δ or 5Δ strain was cultured overnight in YPD medium at a turbidity of OD 600 = 0.01, and 40 μl aliquots were dispensed into 384-well assay-ready plates containing candidate compounds using a liquid dispenser (MultiFlo FX, BioTek).

[0088] After 24 h of incubation at 30°C, the 384-well plate was thoroughly mixed using a vortex mixer (MixMate, Eppendorf) and the OD was measured using a microplate spectrophotometer (Epoch 2, BioTek). 600 was measured.

[0089] 14 In vitro assays to monitor the fate of C-labeled PS were performed as described in Non-Patent Documents 1 and 2. Specifically, PS was resuspended in assay buffer (300 mM sucrose, 20 mM Tris-HCl, pH 7.5, 40 mM KCl, 2 mM CTP, 1 mM S-adenosylmethionine, 0.1 mM MnCl2, 2 mM MgCl2) and diluted with [C100] at a final concentration of 2 μCi / ml. 14 C(U)]-L-serine was added to the yeast heavy membrane fraction (12,000 × g pellet).

[0090] To observe PE methylation, heavy and light membrane fractions (100,000 × g pellet) resuspended in assay buffer (20 mM Tris-HCl, pH 7.5, 300 mM sucrose, 40 mM KCl, 2 mM CTP, 0.5 mM L-serine, 0.1 mM MnCl2, 2 mM MgCl2) were treated with 100 mM ethanol at a final concentration of 15 μCi / ml. 3 H-SAM was added.

[0091] To stop the reaction, 900 μl of 2:1 chloroform / methanol was added to each sample and mixed on a vortex mixer for 15 minutes. Then, 200 μl of 0.1 M KCl and 0.1 M HCl were added to each sample and mixed on a vortex mixer for another 15 minutes at room temperature. After spinning for 5 minutes, the organic phase was collected and dried under N2 gas. The resulting lipids were dissolved in chloroform and analyzed by thin layer chromatography (TLC), followed by radioimaging analysis by an image analyzer (Amersham Typhoon scanner).

[0092] Logarithmically growing yeast cells cultured in SCD medium were observed using a microscope (Model IX83, Olympus) equipped with a confocal unit (CSU-X1, Yokogawa), a 100x objective with a numerical aperture of 1.4 (UPlanSApo, Olympus), and an EM-CCD camera (Evolve512, Photometrics). Images were analyzed using software (MetaMorph, Molecular Devices). GFP was excited using a 488 nm laser (OBIS, Coherent) and its emission was passed through a 520 / 35 nm filter. Sections of confocal fluorescence were collected every 0.2 μm from the top to the bottom of the yeast cells. The resulting confocal images were maximum projected using software (Image J, NIH).

Claims

1. A step of mixing a first microorganism in which the Eki1, Cki1, and Psd2 genes are deleted and a second microorganism in which the genes are not deleted with a candidate substance, respectively, to prepare a medium containing the microorganism and the candidate substance; Growing the first microorganism and the second microorganism in the medium, respectively; and a step of comparing the growth amounts of the first microorganism and the second microorganism, and selecting the candidate substance in the medium containing the candidate substance as a PC synthesis inhibitor when the growth amount of the first microorganism is lower than the growth amount of the second microorganism; A method for screening for a PC synthesis inhibitor, comprising:

2. The screening method according to claim 1 , wherein the first microorganism and the second microorganism are deficient in Pdr1 and / or Pdr3.

3. The method according to claim 1 , wherein the step of examining the amount of proliferation is carried out by measuring the turbidity of the medium.

4. The screening method according to claim 1 , wherein the first microorganism and the second microorganism are each a yeast.

5. A PC synthesis inhibitor comprising one or more compounds of the following formulas (1) to (4): 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 (In the formula, X １ ~X ８ each represents a hydrogen atom, a halogen atom, or a C １ ~C １２ or a chemical group whose terminal group may have a heteroatom selected from the group consisting of a hydroxyl group, a carboxyl group, a sulfonic acid group, a nitro group, and an amino group; n １ ~n ８ are each an integer from 1 to 5, and Y １ and Y ２ each of which may have a hydrogen atom or a heteroatom is １ ~C １２ is a hydrocarbon group.

6. The PC synthesis inhibitor according to claim 5 , which is a bactericide or an antibiotic.

7. The PC synthesis inhibitor according to claim 5, which is a mitochondrial division promoter.

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