WO2022063311A1 - 用于判断线粒体氧化磷酸化通路抑制剂抗癌效果的标志物 - Google Patents

用于判断线粒体氧化磷酸化通路抑制剂抗癌效果的标志物 Download PDF

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WO2022063311A1
WO2022063311A1 PCT/CN2021/121088 CN2021121088W WO2022063311A1 WO 2022063311 A1 WO2022063311 A1 WO 2022063311A1 CN 2021121088 W CN2021121088 W CN 2021121088W WO 2022063311 A1 WO2022063311 A1 WO 2022063311A1
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oxidative phosphorylation
tumor
cells
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PCT/CN2021/121088
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French (fr)
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施裕丰
马文江
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南京施江医药科技有限公司
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Priority to KR1020237013765A priority Critical patent/KR20230078715A/ko
Priority to US18/028,574 priority patent/US20230355647A1/en
Priority to CA3228855A priority patent/CA3228855A1/en
Priority to JP2023543257A priority patent/JP2023544226A/ja
Priority to EP21871680.1A priority patent/EP4218752A4/en
Priority to CN202180006008.7A priority patent/CN114630663A/zh
Publication of WO2022063311A1 publication Critical patent/WO2022063311A1/zh

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Definitions

  • the invention relates to the field of medicine, in particular to a marker for judging the anticancer effect of mitochondrial oxidative phosphorylation pathway inhibitor.
  • Mitochondria are ubiquitous in eukaryotic cells, providing energy and other intermediates necessary for cell growth. Mitochondria, as the energy factory and source of raw materials in cells, are also indispensable organelles for tumor cell tumorigenesis. Inhibiting mitochondrial function can effectively inhibit the occurrence and development of tumors, reduce the malignancy of the patient's tumor, and increase the patient's survival period.
  • Oxidative Phospholytion is one of the most important pathways in mitochondria, which utilizes NADH and FADH derived from pathways such as the tricarboxylic acid cycle and fat oxidation to synthesize ATP.
  • Mitochondrial oxidative phosphorylation pathway consists of more than 90 proteins, these proteins form five protein complexes, complexes I, II, III, IV and V.
  • the first 4 protein complexes also known as the electron transport chain, receive electrons from electron donors NADH and FADH and transfer them to oxygen.
  • Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma. Gboxin is an oxidative phosphorylation inhibitor that can effectively inhibit tumor growth. Nature 567, 341–346 (2019); a study published in "Nature Medicine” found that the inhibitor of mitochondrial oxidative phosphorylation pathway IACS-010759 has a good inhibitory effect on brain tumors and acute myeloid leukemia, see Molina, JR, Sun , Y., Protopopova, M. et al. An inhibitor of oxidative phosphorylation exploits cancer vulnerability. Nat Med 24, 1036–1046 (2016).
  • the object of the present invention is to provide a mitochondrial oxidative phosphorylation pathway expression level or activity, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and / or NNMT gene region DNA CpG site methylation level is used to determine whether tumor patients are suitable for prevention and / or treatment of mitochondrial oxidative phosphorylation pathway inhibitors, so as to carry out precise tumor treatment.
  • Mitochondrial oxidative phosphorylation pathway inhibitors up-regulated mitochondrial oxidative phosphorylation pathway, low expression or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high level of NNMT gene nucleotide site methylation, and/ Or tumors with high levels of DNA CpG methylation in the NNMT gene region have significantly excellent therapeutic effects.
  • the first aspect of the present invention provides the use of an inhibitor of mitochondrial oxidative phosphorylation pathway for preparing a composition or preparation for preventing and/or treating tumors.
  • the tumor is a human tumor.
  • the tumor is a human tumor.
  • the tumor includes a tumor with up-regulated mitochondrial oxidative phosphorylation pathway.
  • the up-regulated mitochondrial oxidative phosphorylation pathway means that the expression level or activity of the mitochondrial oxidative phosphorylation pathway in a certain cell (such as a tumor cell) is greater than that in the same cell or normal cell (such as a tumor cell) Oxidative phosphorylation pathway expression level or activity.
  • the up-regulation of the mitochondrial oxidative phosphorylation pathway includes a high expression level or activity of the mitochondrial oxidative phosphorylation pathway.
  • the up-regulation of mitochondrial oxidative phosphorylation pathway means that the expression level or activity H1 of mitochondrial oxidative phosphorylation pathway of a certain cell (such as tumor cells) is the same as that in the same cell or normal cells (such as paracancerous tissue cells).
  • the same cell refers to a cell with normal expression or activity of mitochondrial oxidative phosphorylation pathway (like a type of tumor cell).
  • the same cell refers to cells of the same species but with normal expression or activity of mitochondrial oxidative phosphorylation pathway.
  • the normal cells refer to normal tissue cells with normal expression or normal activity of mitochondrial oxidative phosphorylation pathway (eg, tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells).
  • mitochondrial oxidative phosphorylation pathway eg, tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells.
  • the tumor includes a tumor with low or no expression of NNMT gene.
  • the NNMT gene is a human NNMT gene.
  • the NNMT gene is a human NNMT gene.
  • the tumor includes a tumor with high DNA methylase expression.
  • the DNA methylase is selected from the group consisting of DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the tumor includes a tumor with high expression of DNMT1.
  • the tumor includes a tumor with high expression of DNMT3a.
  • the tumor includes a tumor with high expression of DNMT3b.
  • the tumor includes a tumor with high UHRF1 expression.
  • the tumor includes a tumor with a high level of methylation at the nucleotide site of the NNMT gene and/or a high level of methylation at the DNA CpG site in the NNMT gene region.
  • the tumor includes a tumor with a high level of methylation at the nucleotide site of the NNMT gene.
  • the tumor includes a tumor with a high level of methylation at the DNA CpG site in the NNMT gene region.
  • the tumor with low expression or no expression of NNMT gene refers to that NNMT protein cannot be detected by NNMT antibody in 1 ⁇ g of protein extracted from the tumor, more preferably 5 ⁇ g, more preferably 10 ⁇ g, More preferably 100 ⁇ g, more preferably 1000 ⁇ g.
  • the tumor with low or no expression of NNMT gene refers to that the expression level of NNMT gene in tumor cells is lower than the expression level of NNMT gene in the same cell or in normal cells (eg, paracancerous tissue cells).
  • the tumor with low or no expression of NNMT gene refers to the difference between the expression level E1 of NNMT gene in tumor cells and the expression level E0 of NNMT gene in the same cell or normal cells (such as adjacent tissue cells) Ratio (E1/E0) ⁇ 1.0.
  • the low expression or non-expression of NNMT gene refers to the expression E1 of NNMT gene in a certain cell (such as tumor cells) and the expression of NNMT gene in the same cell or normal cells (such as adjacent tissue cells)
  • E0 (E1/E0) ⁇ 1.0 preferably ⁇ 0.7, more preferably ⁇ 0.6, more preferably ⁇ 0.5, more preferably ⁇ 0.4, more preferably ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, more preferably ⁇ 0.05, more preferably ⁇ 0.01, more preferably ⁇ 0.005, more preferably ⁇ 0.001, more preferably ⁇ 0.0001, more preferably ⁇ 0.00001, more preferably ⁇ 0.000001, more preferably ⁇ 0.0000001 .
  • the same cell refers to a cell with normal expression of NNMT gene (similar to a type of tumor cell).
  • the same cell refers to cells of the same type but with normal expression of NNMT gene.
  • the normal cells refer to normal tissue cells with normal expression of NNMT gene (such as tumor cell origin cells, tumor adjacent cells or adjacent tumor tissue cells).
  • E0 is the expression level of NNMT gene in cells that normally express NNMT gene.
  • the cells with normal expression of the NNMT gene include cells that are insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the tumor with high expression of DNA methylase refers to that DNA methylase can be detected by DNA methylase antibody detection in 20 ⁇ g of protein extracted from the tumor, more preferably 5 ⁇ g, more preferably 1 ⁇ g, more preferably 0.2 ⁇ g, more preferably 0.05 ⁇ g, more preferably 0.01 ⁇ g.
  • the tumor with high DNA methylase expression refers to that the expression level of DNA methylase in tumor cells is greater than that in the same cell or in normal cells (such as adjacent tissue cells) expression level.
  • the tumor with high expression of DNA methylase refers to the expression level A1 of DNA methylase in tumor cells and DNA methylation in the same cell or normal cells (such as adjacent tissue cells)
  • the same cell refers to a cell that normally expresses DNA methylase (similar to a type of tumor cell).
  • the same cell refers to a cell of the same species but with normal expression of DNA methylase.
  • the normal cells refer to normal tissue cells with normal expression of DNA methylase (eg, tumor cell origin cells, tumor adjacent cells or adjacent tumor tissue cells).
  • DNA methylase eg, tumor cell origin cells, tumor adjacent cells or adjacent tumor tissue cells.
  • A0 is the expression level of DNA methylase in cells that normally express DNA methylase.
  • the cells with normal expression of DNA methylase include cells that are not sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the tumor with high DNMT1 expression refers to that DNMT1 protein can be detected in 20 ⁇ g of protein extracted from the tumor by DNMT1 antibody detection, more preferably 5 ⁇ g, more preferably 1 ⁇ g, more preferably is 0.2 ⁇ g, more preferably 0.05 ⁇ g, more preferably 0.01 ⁇ g.
  • the tumor with high DNMT1 expression refers to that the expression level of DNMT1 in tumor cells is greater than the expression level of DNMT1 in the same cell or normal cells (eg, adjacent tissue cells).
  • the tumor with high DNMT1 expression refers to the ratio of the expression level B1 of DNMT1 in tumor cells to the expression level B0 of DNMT1 in the same cell or normal cells (such as adjacent tissue cells) (B1/B0) >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50.
  • the same cell refers to a cell that normally expresses DNMT1 (similar to a type of tumor cell).
  • the same cell refers to cells of the same species but normally expressing DNMT1.
  • the normal cells refer to normal tissue cells that normally express DNMT1 (such as tumor cell origin cells, tumor adjacent cells or adjacent tumor tissue cells).
  • B0 is the expression level of DNMT1 in cells that normally express DNMT1.
  • the cells that normally express DNMT1 include cells that are not sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the tumor with high DNMT3a expression refers to that DNMT3a protein can be detected in 20 ⁇ g of protein extracted from the tumor by DNMT3a antibody detection, more preferably 5 ⁇ g, more preferably 1 ⁇ g, more preferably is 0.2 ⁇ g, more preferably 0.05 ⁇ g, more preferably 0.01 ⁇ g.
  • the tumor with high DNMT3a expression refers to that the expression level of DNMT3a in tumor cells is greater than the expression level of DNMT3a in the same cell or normal cells (eg, adjacent tissue cells).
  • the tumor with high DNMT3a expression refers to the ratio (C1/C0) of the expression level C1 of DNMT3a in tumor cells to the expression level C0 of DNMT3a in the same cell or normal cells (such as adjacent tissue cells). >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50.
  • the same cell refers to a cell that normally expresses DNMT3a (similar to a type of tumor cell).
  • the same cell refers to cells of the same species but normally expressing DNMT3a.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) in which DNMT3a is normally expressed.
  • C0 is the expression level of DNMT3a in cells that normally express DNMT3a.
  • the cells that normally express DNMT3a include cells that are insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the tumor with high DNMT3b expression refers to that DNMT3b protein can be detected in 20 ⁇ g of protein extracted from the tumor by DNMT3b antibody detection, more preferably 5 ⁇ g, more preferably 1 ⁇ g, more preferably is 0.2 ⁇ g, more preferably 0.05 ⁇ g, more preferably 0.01 ⁇ g.
  • the tumor with high DNMT3b expression refers to that the expression level of DNMT3b in tumor cells is greater than the expression level of DNMT3b in the same cell or in normal cells (eg, paracancerous tissue cells).
  • the tumor with high DNMT3b expression refers to the ratio (D1/D0) of the expression level D1 of DNMT3b in tumor cells to the expression level D0 of DNMT3b in the same cell or in normal cells (such as paracancerous tissue cells). >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50.
  • the same cell refers to a cell that normally expresses DNMT3b (similar to a type of tumor cell).
  • the same cell refers to cells of the same species but normally expressing DNMT3b.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) in which DNMT3b is normally expressed.
  • D0 is the expression level of DNMT3b in cells that normally express DNMT3b.
  • the cells that normally express DNMT3b include cells that are insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the tumor with high expression of UHRF1 means that UHRF1 protein can be detected in 20 ⁇ g of protein extracted from the tumor by UHRF1 antibody detection, more preferably 5 ⁇ g, more preferably 1 ⁇ g, more preferably is 0.2 ⁇ g, more preferably 0.05 ⁇ g, more preferably 0.01 ⁇ g.
  • the tumor with high UHRF1 expression refers to that the expression level of UHRF1 in tumor cells is greater than the expression level of UHRF1 in the same cell or in normal cells (eg, paracancerous tissue cells).
  • the tumor with high UHRF1 expression refers to the ratio (F1/F0) of the UHRF1 expression level F1 in tumor cells to the UHRF1 expression level F0 in the same cell or in normal cells (such as paracancerous tissue cells). >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50.
  • the same cell refers to a cell that normally expresses UHRF1 (similar to a type of tumor cell).
  • the same cell refers to cells of the same type but normally expressing UHRF1.
  • the normal cells refer to normal tissue cells that normally express UHRF1 (such as tumor cell origin cells, tumor adjacent cells or adjacent tumor tissue cells).
  • F0 is the expression level of UHRF1 in cells that normally express UHRF1.
  • the cells that normally express UHRF1 include cells that are not sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the high methylation level of the NNMT gene nucleotide site means that the methylation level of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is greater than that of the same cell or normal cell ( For example, the methylation level of NNMT gene nucleotide sites in adjacent tissue cells).
  • the high methylation level of the NNMT gene nucleotide site means that the methylation level L1 of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is the same as that of the same cell or normal cell.
  • the high methylation level of the NNMT gene nucleotide site means that the methylation level of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is greater than or equal to 1%, preferably ⁇ 3%, preferably ⁇ 5%, preferably ⁇ 10%, preferably ⁇ 15%, preferably ⁇ 20%, more preferably ⁇ 25%, more preferably ⁇ 30%, more preferably ⁇ 40%, more preferably ⁇ 50%.
  • the same cell refers to a cell with a normal level of methylation at the nucleotide site of the NNMT gene (similar to a type of tumor cell).
  • the same cell refers to cells of the same species but with a normal level of methylation at the nucleotide site of the NNMT gene.
  • the normal cells refer to normal tissue cells with a normal level of methylation at NNMT gene nucleotide sites (eg, tumor cell origin cells, tumor adjacent cells or adjacent tumor tissue cells).
  • the cells with a normal level of methylation at the nucleotide site of the NNMT gene include cells that are insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the high methylation level of the NNMT gene nucleotide site means that the methylation level (M%) of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is greater than or equal to 3% and less than or equal to M1%, where M1 is any positive integer between 3 and 100.
  • M1 is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or 100.
  • the methylation level of the nucleotide site of the NNMT gene refers to the ratio of the number of methylated nucleotides in the NNMT gene region to the number of all nucleotides in the NNMT gene region.
  • the methylation level of the nucleotide site of the NNMT gene includes the methylation level of the nucleotide site of the promoter region of the NNMT gene.
  • nucleotide sequence of the NNMT gene promoter region is shown in SEQ ID NO: 1.
  • the methylation level of the nucleotide site of the NNMT gene includes the methylation level of the nucleotide site within the region from 1050 bp before the transcription start site to 499 bp after the transcription start site of the NNMT gene .
  • 1050bp before the transcription initiation site of the NNMT gene to 499bp after the transcription initiation site are positions 951-2500 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the methylation level of the NNMT gene nucleotide site includes the methylation of nucleotide sites in the region from 1050 bp before the transcription start site of the NNMT gene to 193 bp before the transcription start site of the gene level.
  • the 1050 bp before the transcription start site of the NNMT gene to the 193 bp before the transcription start site of the gene are positions 951-1808 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the methylation level of the nucleotide site of the NNMT gene includes the methylation level of the nucleotide site within the region from 840 bp before the transcription start site to 469 bp before the transcription start site of the NNMT gene .
  • the 840 bp before the transcription start site of the NNMT gene to the 469 bp before the transcription start site are positions 1161-1532 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the methylation level of the NNMT gene nucleotide site includes any two positions of human chromosome 11 at positions 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 The level of methylation at nucleotide sites within the region between the sites, including the two sites themselves.
  • the methylation level of the NNMT gene nucleotide site comprises one or more of the 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 positions of human chromosome 11.
  • the methylation level of the NNMT gene nucleotide site includes the nucleotide methylation level of a site selected from the group consisting of: human chromosome 11 position 114165695, human chromosome 11 position 114165730 , human chromosome 11 at position 114165769, human chromosome 11 at position 114165804, human chromosome 11 at position 114165938, human chromosome 11 at position 114166050, human chromosome 11 at position 114166066, or a combination thereof.
  • the methylation level of the NNMT gene nucleotide site includes the 1161st, 1196th, 1235th, 1270th, Methylation levels of nucleotide sites in the region between any two of positions 1404, 1516 and 1532, including the two sites themselves.
  • the methylation level of the NNMT gene nucleotide site includes the 1161st, 1196th, 1235th, 1270th, 1270th, Nucleotide methylation levels at one or more of positions 1404, 1516, and 1532 (eg, 2, 3, 4, 5, 6, or 7).
  • the methylation level of the DNA CpG site in the NNMT gene region comprises a nucleotide methylation level selected from the following sequence sites of SEQ ID NO: 1: the 1161st position, the 1196th position, bit 1235, bit 1270, bit 1404, bit 1516, bit 1532, or a combination thereof.
  • the high methylation level of the DNA CpG site in the NNMT gene region means that the methylation level of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) is greater than that of the same cell or normal cell ( The methylation level of DNA CpG sites in the NNMT gene region in paracancerous tissue cells).
  • the high methylation level of the DNA CpG site in the NNMT gene region means that the methylation level W1 of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) is the same as that of the same cell or normal cell.
  • the high methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) ⁇ 1%, preferably ⁇ 3%, preferably ⁇ 5%, preferably ⁇ 10%, preferably ⁇ 15%, preferably ⁇ 20%, more preferably ⁇ 25%, more preferably ⁇ 30%, more preferably ⁇ 40%, more preferably ⁇ 50%.
  • the same cell refers to a cell with a normal level of methylation at the DNA CpG site in the NNMT gene region (similar to a type of tumor cell).
  • the same cell refers to cells of the same species but with a normal level of methylation at the DNA CpG site in the NNMT gene region.
  • the normal cells refer to normal tissue cells with normal levels of methylation at the DNA CpG site in the NNMT gene region (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells).
  • the cells with normal methylation levels of DNA CpG sites in the NNMT gene region include cells that are not sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the high methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level (M%) of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) ⁇ 3% and less than or equal to M2%, where M2 is any positive integer between 3 and 100.
  • M2 is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or 100.
  • the CpG site methylation level refers to the ratio of the number of methylated CpG nucleotides in a certain gene region to the number of all nucleotides in the gene region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG nucleotides in the NNMT gene region to the number of all nucleotides in the NNMT gene region.
  • the CpG site methylation level refers to the ratio of the number of methylated CpG nucleotides in a certain gene region to the number of all CpG nucleotides in the gene region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG nucleotides in the NNMT gene region to the number of all CpG nucleotides in the NNMT gene region.
  • the DNA CpG site methylation level refers to the ratio of the number of methylated CpG sites in DNA in a certain region to the total number of CpG sites in the DNA in this region.
  • the methylation level of the DNA CpG site refers to the ratio of the number of methylated CpG nucleotides in the DNA of a certain region to the number of all nucleotides in the DNA in the region.
  • the methylation level of DNA CpG sites refers to the ratio of the number of methylated CpG nucleotides in DNA in a certain region to the total number of CpG nucleotides in DNA in that region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG sites in the DNA of the NNMT gene region to the total number of CpG sites in the DNA in the NNMT gene region.
  • the methylation level of the DNA CpG site in the NNMT gene region refers to the number of methylated CpG nucleotides in the DNA in the NNMT gene region accounting for the total number of CpG nucleotides in the DNA in the NNMT gene region. ratio.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the promoter region of the NNMT gene.
  • nucleotide sequence of the NNMT gene promoter region is shown in SEQ ID NO: 1.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the region from 1050bp before the transcription start site of the NNMT gene to 499bp after the transcription start site.
  • 1050bp before the transcription initiation site of the NNMT gene to 499bp after the transcription initiation site are positions 951-2500 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the region from 1050 bp before the transcription start site of the NNMT gene to 193 bp before the transcription start site of the gene.
  • the 1050 bp before the transcription start site of the NNMT gene to the 193 bp before the transcription start site of the gene are positions 951-1808 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the DNA CpG site methylation level in the NNMT gene region includes the DNA CpG site methylation level in the region from 840 bp before the transcription start site of the NNMT gene to 469 bp before the transcription start site.
  • the 840 bp before the transcription start site of the NNMT gene to the 469 bp before the transcription start site are positions 1161-1532 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the methylation level of the DNA CpG site in the NNMT gene region includes any two of positions 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 of human chromosome 11 DNA CpG site methylation levels within the region between the sites, including the two sites themselves.
  • the methylation level of the DNA CpG site in the NNMT gene region includes one or more of positions 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 of human chromosome 11 methylation level at each (eg, 2, 3, 4, 5, 6, or 7) sites.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of a site selected from the group consisting of: human chromosome 11 position 114165695, human chromosome 11 position 114165730, human chromosome 11 position 114165730 chromosome 114165769, human chromosome 11 position 114165804, human chromosome 11 position 114165938, human chromosome 11 position 114166050, human chromosome 11 position 114166066, or a combination thereof.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the 1161st, 1196th, 1235th, 1270th, 1270th, DNA CpG site methylation levels in the region between any two of positions 1404, 1516 and 1532, including the two sites themselves.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the 1161st, 1196th, 1235th, 1270th, 1270th, Methylation level at one or more of positions 1404, 1516 and 1532 (eg 2, 3, 4, 5, 6 or 7).
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the sequence site of SEQ ID NO: 1 selected from the group consisting of: No. 1161, No. 1196, No. 1235 bit, bit 1270, bit 1404, bit 1516, bit 1532, or a combination thereof.
  • the tumor is selected from the group consisting of lung cancer, kidney cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, lymphoma, leukemia, pancreatic cancer, brain tumor, liver cancer, prostate cancer, melanoma, or a combination thereof.
  • the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, metastatic lung cancer, or a combination thereof.
  • the colon cancer includes colon adenocarcinoma.
  • the rectal cancer includes rectal adenocarcinoma.
  • the colorectal cancer includes colorectal adenocarcinoma.
  • the lymphoma is selected from the group consisting of B-cell lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, large cell lymphoma, histiocytic lymphoma, or a combination thereof.
  • the lymphoma includes diffuse large B-cell lymphoma.
  • the brain tumor is selected from the group consisting of glioblastoma, glioma, or a combination thereof.
  • the glioblastoma includes glioblastoma multiforme.
  • the brain tumor includes medulloblastoma.
  • the renal cancer is selected from the group consisting of renal clear cell adenocarcinoma, metastatic renal cancer, or a combination thereof.
  • kidney cancer cells include kidney cancer Wilms cells.
  • the leukemia is selected from the group consisting of T lymphocytic leukemia, myeloid leukemia, or a combination thereof.
  • the T-lymphocytic leukemia includes acute T-lymphocytic leukemia.
  • the myeloid leukemia includes acute myeloid leukemia.
  • the myeloid leukemia includes AML acute myeloid leukemia.
  • the myeloid leukemia includes M4 grade AML acute myeloid leukemia.
  • the myeloid leukemia includes FAB M4 grade AML acute myeloid leukemia
  • the expression includes protein expression and/or mRNA expression.
  • the prostate cancer is selected from the group consisting of metastatic prostate cancer.
  • the metastatic prostate cancer is selected from the group consisting of brain metastatic prostate cancer, bone metastatic prostate cancer, or a combination thereof.
  • the breast cancer is selected from the group consisting of breast ductal carcinoma, metastatic breast cancer, or a combination thereof.
  • the breast ductal carcinoma includes primary breast ductal carcinoma.
  • the breast ductal carcinoma includes grade 3 primary breast ductal carcinoma.
  • the pancreatic cancer includes liver metastatic pancreatic cancer.
  • the mitochondrial oxidative phosphorylation pathway inhibitor comprises the compound of formula I, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof;
  • R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxy, mercapto, amino, substituted or unsubstituted C1-C12 alkyl, substituted or Unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl, substituted or unsubstituted C1-C12 alkoxy, substituted or unsubstituted C1-C12 alkylthio, substituted or Unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12 membered heteroaryl;
  • R 5 is none, hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted 3-12-membered heterocycloalkane base, substituted or unsubstituted C1-C12 alkoxy, substituted or unsubstituted C1-C12 alkylthio, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12-membered heteroaryl;
  • any "substitute” refers to that one or more (preferably 1, 2, 3, or 4) hydrogen atoms on the group are replaced by a substituent selected from the group consisting of: C1-C8 alkanes base, C3-C8 cycloalkyl, C1-C8 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C8 alkoxy base, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10-membered hetero Aryl, methanesulfonyl, sulfonyl;
  • the heterocyclic ring of the heterocycloalkyl group and the heteroaryl group each independently has 1-4 (preferably 1, 2, 3 or 4) heteroatoms selected from N, O and S.
  • R5 is none, is a double bond.
  • R5 is not none, is a single key.
  • R5 is not none and is a double bond, and the N atom connected to R5 is N + .
  • R5 is none, hydrogen or C1-C3 alkyl.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted Substituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted Substituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C6-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted Substituted C1-C6 alkyl, substituted or unsubstituted C5-C8 cycloalkyl, substituted or unsubstituted 5-8 membered heterocycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C6-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted Substituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkylthio, substituted or unsubstituted C6-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted Substituted C1-C4 alkyl, substituted or unsubstituted C5-C8 cycloalkyl, substituted or unsubstituted 5-8 membered heterocycloalkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkylthio, substituted or unsubstituted C6-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted Substituted C1-C4 alkyl, substituted or unsubstituted C5-C8 cycloalkyl, substituted or unsubstituted 5-8 membered heterocycloalkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkylthio, substituted or unsubstituted C6 aryl, substituted or unsubstituted C7 aryl, substituted or unsubstituted C8 aryl, substituted or unsubstituted 5-8 membered (such as 5, 6, 7, 8) Heteroaryl.
  • R 1 , R 2 , R 3 , R 4 , R 7 and R 8 are each independently hydrogen.
  • R 5 is hydrogen, methyl, ethyl, propyl, or butyl.
  • R 6 is hydrogen, methyl, ethyl, propyl, butyl, phenyl, trifluoromethyl-phenyl-.
  • trifluoromethyl-phenyl is mono-substituted trifluoromethyl-phenyl-.
  • the trifluoromethyl group is substituted at the ortho, meta or para position of the benzene ring.
  • trifluoromethyl-phenyl is:
  • R 6 is hydrogen, methyl, ethyl, propyl, butyl, unsubstituted phenyl, or substituted phenyl.
  • the substituted phenyl group means that one or more (eg 2, 3 or 4) hydrogens of the phenyl group are substituted by trifluoromethyl.
  • the substituted phenyl group means that one or more (eg 2, 3 or 4) hydrogens of the phenyl group are substituted by trifluoromethyl.
  • the substituted phenyl group means that one hydrogen of the phenyl group is substituted by a trifluoromethyl group.
  • the substituted phenyl group means that one of the ortho, meta or para hydrogens of the phenyl group is substituted with a trifluoromethyl group.
  • R 6 is hydrogen, methyl, ethyl, propyl, butyl, or
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl (such as trifluoromethyl), C3- C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio , C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10-membered heteroaryl.
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl (such as trifluoromethyl) base), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C6 alkoxy, C1-C6 alkylthio, C3-C8 cycloalkoxy, C3- C8 cycloalkylthio, C1-C6 haloalkoxy, C1-C6 haloalkylthio, C6-C10 aryl, 5-8 membered heteroaryl.
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, C1-C4 alkyl, C3-C8 cycloalkyl, C1-C4 haloalkyl (such as trifluoromethyl) base), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C4 alkoxy, C1-C6 alkylthio, C3-C8 cycloalkoxy, C3- C8 cycloalkylthio, C1-C4 haloalkoxy, C1-C4 haloalkylthio, C6-C10 aryl, 5-8 membered heteroaryl.
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, C1-C4 haloalkyl (eg, trifluoromethyl).
  • R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen or trifluoromethyl.
  • R 10 , R 11 , R 12 and R 14 are each independently hydrogen.
  • R 13 is trifluoromethyl.
  • Z 1 is
  • Z 1 is
  • R 9 is a substituted or unsubstituted cyclohexyl group.
  • the substituted cyclohexyl group means that one or more (eg 2, 3 or 4) hydrogens of the cyclohexyl group are each independently substituted by a C1-C4 alkyl group.
  • the substituted cyclohexyl group means that one or more (eg 2, 3 or 4) hydrogens of the cyclohexyl group are each independently substituted with methyl, ethyl, propyl and butyl.
  • the substituted cyclohexyl group means that the hydrogens at the 1-position and the 4-position of the cyclohexyl group are substituted by C1-C4 alkyl groups.
  • the substituted cyclohexyl group means that the hydrogens at the 1-position and the 4-position of the cyclohexyl group are substituted by methyl, ethyl, propyl, and butyl.
  • R 9 is 1-propyl-4-methyl-cyclohexyl-.
  • R 9 is 1-isopropyl-4-methyl-cyclohexyl-.
  • R 9 is
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 and R 24 are each independently hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10 membered heteroaryl.
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 and R 24 are each independently hydrogen, C1-C6 alkyl, C3- C8 cycloalkyl, C1-C6 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C6 alkoxy, C1- C6 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C6 haloalkoxy, C1-C6 haloalkylthio, C6-C10 aryl, 5-10 membered heteroaryl.
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 and R 24 are each independently hydrogen, C1-C4 alkyl, C3- C8 cycloalkyl, C1-C4 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C4 alkoxy, C1- C4 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C4 haloalkoxy, C1-C4 haloalkylthio, C6-C10 aryl, 5-10 membered heteroaryl.
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 and R 24 are each independently hydrogen, methyl, ethyl, propyl , Butyl.
  • the propyl group is isopropyl.
  • R 9 is
  • R 16 , R 17 , R 18 , R 19 , R 20 , R 22 , R 23 and R 24 are as defined above.
  • R 9 is
  • R 9 is
  • the heterocycles of the heterocycloalkyl and heteroaryl groups each independently have 1-4 (preferably 1, 2, 3 or 4) selected from N, O and S. of heteroatoms.
  • R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 and Z1 are as defined above.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and Z1 are as defined above.
  • the compound of formula I has the following structure of formula I-3:
  • the mitochondrial oxidative phosphorylation pathway inhibitor comprises the compound of formula II, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof;
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 and R 36 are each independently hydrogen, halogen, hydroxyl, mercapto, amino, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted 3-12 membered heterocycloalkyl, substituted or unsubstituted C1-C12 alkoxy, substituted or Unsubstituted C1-C12 alkylthio, substituted or unsubstituted C1-C12 haloalkoxy, substituted or unsubstituted C1-C12 haloalkylthio, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-12-membered heteroaryl;
  • Z 2 and Z 3 are each independently a substituted or unsubstituted C6-C12 arylene group, a substituted or unsubstituted 3-12-membered heteroarylene group;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;
  • substitution means that one or more (preferably 1, 2, 3, or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group: C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10 membered heteroaryl , methanesulfonyl, sulfonyl;
  • heterocycles of the heterocycloalkyl, heteroaryl, arylene and heteroarylene independently have 1-4 (preferably 1, 2, 3 or 4) selected from N, O and S heteroatoms.
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 and R 36 are each independently hydrogen, halogen, Hydroxyl, mercapto, amino, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted C1- C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 haloalkoxy, substituted or unsubstituted C1-C10 haloalkylthio, substituted or unsubstituted C6-C10 aryl group, substituted or unsubstituted 5-10 membered heteroaryl.
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 and R 36 are each independently hydrogen, halogen, Hydroxyl, mercapto, amino, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted C1- C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C1-C8 haloalkoxy, substituted or unsubstituted C1-C8 haloalkylthio, substituted or unsubstituted C6-C10 aryl group, substituted or unsubstituted 5-10 membered heteroaryl.
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 and R 36 are each independently hydrogen, halogen, Hydroxyl, mercapto, amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted C1- C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C1-C6 haloalkoxy, substituted or unsubstituted C1-C6 haloalkylthio, substituted or unsubstituted C6-C10 aryl group, substituted or unsubstituted 5-10 membered heteroaryl.
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 and R 36 are each independently hydrogen, halogen, Hydroxyl, mercapto, amino, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted C1- C4 alkoxy, substituted or unsubstituted C1-C4 alkylthio, substituted or unsubstituted C1-C4 haloalkoxy, substituted or unsubstituted C1-C4 haloalkylthio, substituted or unsubstituted C6-C8 aryl group, substituted or unsubstituted 5-8 membered heteroaryl.
  • R 25 , R 26 , R 28 , R 29 , R 30 , R 31 , R 32 , R 34 , R 35 and R 36 are each independently hydrogen.
  • R 27 is substituted or unsubstituted C1-C4 haloalkoxy, substituted or unsubstituted C1-C4 haloalkylthio.
  • R 27 is substituted or unsubstituted C1-C3 haloalkoxy, substituted or unsubstituted C1-C3 haloalkylthio.
  • R 27 is substituted or unsubstituted C1-C2 haloalkoxy, substituted or unsubstituted C1-C2 haloalkylthio.
  • R 27 is trifluoromethyl-O-, trifluoromethyl-S-,
  • R 33 is a substituted or unsubstituted 3-10 membered (eg 5, 6, 7, 8, 9, 10) membered heterocycloalkyl.
  • the heterocycloalkyl group is a fully saturated heterocycloalkyl group.
  • R 33 is substituted or unsubstituted hexahydropyridyl.
  • R 33 is a substituted or unsubstituted hexahydropyridyl group, and the substitution refers to one or more (eg 2, 3, 4, 5 or 6) hydrogens of the hexahydropyridyl group each independently substituted with a substituent selected from the group consisting of methylsulfonyl, sulfonyl.
  • R 33 is
  • R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 , R 44 , R 45 and R 46 are each independently hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, methanesulfonic acid Acyl, sulfonyl.
  • R 37 , R 38 , R 39 , R 40 , R 41 , R 43 , R 44 , R 45 and R 46 are each independently hydrogen.
  • R 42 is methanesulfonyl or sulfonyl.
  • n 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • n 1
  • Z 2 and Z 3 are each independently a substituted or unsubstituted C6-C10 arylene group, a substituted or unsubstituted 3-10-membered heteroarylene group.
  • Z 2 and Z 3 are each independently a substituted or unsubstituted C6-C8 arylene group, a substituted or unsubstituted 3-8 membered heteroarylene group.
  • Z 2 and Z 3 are each independently a substituted or unsubstituted C6-C8 arylene group, a substituted or unsubstituted 3-7 membered heteroarylene group.
  • Z 2 and Z 3 are each independently substituted or unsubstituted C6 arylene, substituted or unsubstituted C7 arylene, substituted or unsubstituted C8 arylene, substituted or unsubstituted 3-membered heteroarylene, substituted or unsubstituted 4-membered heteroarylene, substituted or unsubstituted 5-membered heteroarylene, substituted or unsubstituted 6-membered heteroarylene, substituted or unsubstituted 7-membered heteroarylene Membered heteroarylene, substituted or unsubstituted 8 membered heteroarylene, substituted or unsubstituted 9 membered heteroarylene, substituted or unsubstituted 10 membered heteroarylene.
  • Z 2 and Z 3 are each independently phenylene, substituted or unsubstituted oxadiazolyl, and substituted or unsubstituted triazolyl.
  • Z 2 is a substituted or unsubstituted oxadiazolyl group.
  • the oxadiazolylide group is 1,2,4-oxadiazolylidene
  • the triazolylidene group is a 1H-1,2,4-triazolylidene group.
  • Z 2 and Z 3 are each independently
  • R 47 is hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl.
  • R 47 is hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl.
  • R 47 is hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl.
  • R 47 is hydrogen, C1-C4 alkyl, C3-C8 cycloalkyl.
  • R 47 is hydrogen, C1-C2 alkyl, C3-C8 cycloalkyl.
  • R 47 is hydrogen, methyl, ethyl, propyl, or butyl.
  • Z 2 is
  • Z 3 is wherein R 47 is as defined above.
  • heterocycles of the heterocycloalkyl, heteroaryl, arylene and heteroarylene each independently have 1-4 (preferably 1, 2, 3 or 4 ) heteroatoms selected from N, O and S.
  • the compound of formula II has the following structure of formula II-1:
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 47 and n are as defined above.
  • the mitochondrial oxidative phosphorylation pathway inhibitor comprises the compound of formula III, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof;
  • R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , R 72 , R 73 , R 74 , R 75 , R 76 , R 77 , R 78 , R 79 , R 80 , R 81 , R 82 , R 83 , R 84 , R 85 , R 86 , R 87 , R 88 , R 89 , R 90 and R 91 are each independently hydrogen, halogen, hydroxy, hydroxy-(C1-C12 alkyl) -, mercapto, amino, substituted or unsubstituted C1-C12 alkyl, substituted
  • substitution means that one or more (preferably 1, 2, 3, or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group: C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10 membered heteroaryl , methanesulfonyl, sulfonyl;
  • the heterocyclic ring of the heterocycloalkyl group and the heteroaryl group each independently has 1-4 (preferably 1, 2, 3 or 4) heteroatoms selected from N, O and S.
  • R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , R 72 , R 73 , R 74 , R 75 , R 76 , R 77 , R 78 , R 79 , R 80 , R 81 , R 82 , R 83 , R 84 , R 85 , R 86 , R 87 , R 88 , R 89 , R 90 and R 91 are each independently hydrogen, halogen, hydroxy, hydroxy- (C1-C10 alkyl)-, mercapto, amino, substituted or unsubstituted C1-C10 alkyl
  • R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , R 72 , R 73 , R 74 , R 75 , R 76 , R 77 , R 78 , R 79 , R 80 , R 81 , R 82 , R 83 , R 84 , R 85 , R 86 , R 87 , R 88 , R 89 , R 90 and R 91 are each independently hydrogen, halogen, hydroxy, hydroxy- (C1-C8 alkyl)-, mercapto, amino, substituted or unsubstituted C1-C8 alkyl
  • R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , R 72 , R 73 , R 74 , R 75 , R 76 , R 77 , R 78 , R 79 , R 80 , R 81 , R 82 , R 83 , R 84 , R 85 , R 86 , R 87 , R 88 , R 89 , R 90 and R 91 are each independently hydrogen, halogen, hydroxy, hydroxy- (C1-C6 alkyl)-, mercapto, amino, substituted or unsubstituted C1-C6 alkyl
  • R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , R 72 , R 73 , R 74 , R 75 , R 76 , R 77 , R 78 , R 79 , R 80 , R 81 , R 82 , R 83 , R 84 , R 85 , R 86 , R 87 , R 88 , R 89 , R 90 and R 91 are each independently hydrogen, hydroxyl, hydroxyl-(C1 -C4 alkyl)-, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstit
  • R 48 , R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 55 , R 56 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , R 72 , R 73 , R 74 , R 75 , R 76 , R 77 , R 78 , R 79 , R 80 , R 81 , R 82 , R 83 , R 84 , R 85 , R 86 , R 87 , R 88 , R 89 , R 90 and R 91 are each independently hydrogen, methyl, ethyl, propyl, butyl, hydroxy-propyl-, mercapto-propyl-, hydroxy, mercapto.
  • hydroxy-propyl- is monohydroxy-propyl-.
  • mercapto-propyl- is monomercapto-propyl-.
  • any "substituted” refers to that one or more (preferably 1, 2, 3, or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group : C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C6 alkoxy, C1-C6 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C6 haloalkoxy, C1-C6 haloalkylthio, C6-C10 aryl, 5-10 membered heteroaryl, methylsulfonyl, sulfonyl.
  • a substituent selected from the following group : C1-C6 alkyl, C3-C
  • any "substituted” refers to that one or more (preferably 1, 2, 3, or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group : C1-C4 alkyl, C3-C8 cycloalkyl, C1-C4 haloalkyl (such as trifluoromethyl), C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C4 alkoxy, C1-C4 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio, C1-C4 haloalkoxy, C1-C4 haloalkylthio, C6-C10 aryl, 5-10 membered heteroaryl, methylsulfonyl, sulfonyl.
  • a substituent selected from the following group : C1-C4 alkyl, C3-C
  • the heterocycles of the heterocycloalkyl and heteroaryl groups each independently have 1-4 (preferably 1, 2, 3 or 4) selected from N, O and S. of heteroatoms.
  • the mitochondrial oxidative phosphorylation pathway inhibitor is selected from the following group:
  • the mitochondrial oxidative phosphorylation pathway inhibitor is selected from the following group:
  • the composition is a pharmaceutical composition.
  • composition or preparation further includes a pharmaceutically acceptable carrier.
  • the expression is mRNA or protein expression.
  • the dosage form of the composition or preparation is a solid preparation, a liquid preparation or a semi-solid preparation.
  • the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation
  • the dosage form of the composition or preparation is tablet, injection, infusion, ointment, gel, solution, microsphere or film.
  • the second aspect of the present invention provides a marker for judging whether a tumor patient is suitable for prevention and/or treatment with an inhibitor of mitochondrial oxidative phosphorylation pathway, the marker includes the expression level or activity of mitochondrial oxidative phosphorylation pathway, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level.
  • the present invention also provides the use of the marker (or its expression level, or activity or methylation level) or its detection reagent, which is used to prepare a kit for diagnosis of tumor patients Whether it is appropriate to use inhibitors of the mitochondrial oxidative phosphorylation pathway for prophylaxis and/or treatment.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the promoter region of the NNMT gene.
  • the NNMT gene when the mitochondrial oxidative phosphorylation pathway is up-regulated, the NNMT gene is under-expressed or not expressed, the DNA methylase is over-expressed, the UHRF1 is over-expressed, the NNMT gene nucleotide site methyl
  • the tumor patients are suitable for prevention and/or treatment with inhibitors of mitochondrial oxidative phosphorylation pathway.
  • the NNMT gene when the mitochondrial oxidative phosphorylation pathway is down-regulated, the NNMT gene is overexpressed, the DNA methylase is under-expressed, the UHRF1 is under-expressed, and the NNMT gene nucleotide site methylation level is low in the tumor cells of the tumor patient , and/or the low methylation level of DNA CpG sites in the NNMT gene region, the tumor patient is not suitable for prevention and/or treatment of mitochondrial oxidative phosphorylation pathway inhibitors.
  • the tumor patient is suitable for the mitochondrial oxidative phosphorylation pathway inhibitor, including the tumor patient's tumor is sensitive to the mitochondrial oxidative phosphorylation pathway inhibitor.
  • the tumor patient is not suitable for the mitochondrial oxidative phosphorylation pathway inhibitor, including the tumor patient's tumor is not sensitive to the mitochondrial oxidative phosphorylation pathway inhibitor.
  • the DNA methylase is selected from the group consisting of DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the tumor with up-regulated mitochondrial oxidative phosphorylation pathway is as described in the first aspect of the present invention.
  • the tumor with low or no expression of the NNMT gene is as described in the first aspect of the present invention.
  • the tumor with high expression of DNA methylase (eg DNMT1) is as described in the first aspect of the present invention.
  • the tumor with high UHRF1 expression is as described in the first aspect of the present invention.
  • the tumor with high methylation level of the NNMT gene nucleotide site is as described in the first aspect of the present invention.
  • the tumor with high methylation level of DNA CpG site in the NNMT gene region is as described in the first aspect of the present invention.
  • the down-regulation of mitochondrial oxidative phosphorylation pathway means that the expression level or activity H1 of mitochondrial oxidative phosphorylation pathway in a certain cell (such as tumor cells) is the same as that in the same cell or normal cells (such as paracancerous tissue cells).
  • the high expression of NNMT gene refers to the expression E1 of NNMT gene in a certain cell (such as tumor cells) and the expression E0 of NNMT gene in the same cell or normal cells (such as adjacent tissue cells)
  • the tumor with low expression of DNA methylase refers to the expression level A1 of DNA methylase in tumor cells and DNA methylation in the same cell or in normal cells (such as adjacent tissue cells)
  • the tumor with low UHRF1 expression refers to the ratio (F1/F0) of UHRF1 expression level F1 in tumor cells to the UHRF1 expression level F0 in the same cell or in normal cells (such as paracancerous tissue cells).
  • ⁇ 1.0 preferably ⁇ 0.7, more preferably ⁇ 0.6, more preferably ⁇ 0.5, more preferably ⁇ 0.4, more preferably ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, more preferably ⁇ 0.05 , more preferably ⁇ 0.01, more preferably ⁇ 0.005, more preferably ⁇ 0.001, more preferably ⁇ 0.0001, more preferably ⁇ 0.00001, more preferably ⁇ 0.000001, more preferably ⁇ 0.0000001.
  • the low methylation level of the NNMT gene nucleotide site means that the methylation level L1 of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is the same cell or normal cell.
  • the ratio (L1/L0) of methylation level L0 of NNMT gene nucleotide sites in (such as adjacent tissue cells) ⁇ 1.0, preferably ⁇ 0.7, more preferably ⁇ 0.6, more preferably ⁇ 0.5, better ⁇ 0.4, more preferably ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, more preferably ⁇ 0.05, more preferably ⁇ 0.01, more preferably ⁇ 0.005, more preferably ⁇ 0.001, more preferably ⁇ 0.0001, more preferably ⁇ 0.00001, more preferably ⁇ 0.000001, more preferably ⁇ 0.0000001.
  • the low methylation level of the DNA CpG site in the NNMT gene region means that the methylation level W1 of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) is the same cell or normal cell.
  • the ratio of methylation level W0 of DNA CpG sites in the NNMT gene region (W1/W0) in the NNMT gene region (such as adjacent tissue cells) ⁇ 1.0, preferably ⁇ 0.7, more preferably ⁇ 0.6, more preferably ⁇ 0.5, better ⁇ 0.4, more preferably ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, more preferably ⁇ 0.05, more preferably ⁇ 0.01, more preferably ⁇ 0.005, more preferably ⁇ 0.001, more preferably ⁇ 0.0001, more preferably ⁇ 0.00001, more preferably ⁇ 0.000001, more preferably ⁇ 0.0000001.
  • a detection kit in a third aspect of the present invention, includes:
  • NNMT gene expression level For detecting mitochondrial oxidative phosphorylation pathway expression level or activity, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene Detection reagent for methylation level of DNA CpG sites.
  • the detection sample of the detection kit includes tumor cells.
  • NNMT gene expression refers to the expression of the gene mRNA and the protein
  • the methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level of the DNA CpG site in the promoter region of the NNMT gene.
  • the methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level of the DNA CpG site in the region from 1050 bp before the transcription initiation site of the NNMT gene to 499 bp after the transcription initiation site of the gene.
  • the methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level of the DNA CpG site in the region from 1050 bp before the transcription start site of the NNMT gene to 193 bp before the gene transcription start site.
  • the methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level of the DNA CpG site in the region from 840 bp before the transcription start site to 469 bp before the transcription start site of the NNMT gene.
  • the fourth aspect of the present invention provides the use of the detection kit according to the third aspect of the present invention, for preparing a companion diagnostic kit for judging whether a tumor patient is suitable for adopting mitochondrial oxidative phosphorylation pathway inhibitors for prophylaxis and/or treatment.
  • the companion diagnostic kit further includes instructions or labels
  • the NNMT gene When the mitochondrial oxidative phosphorylation pathway is upregulated, the NNMT gene is underexpressed or not expressed, the DNA methylase is overexpressed, the UHRF1 is overexpressed, the NNMT gene nucleotide site methylation level is high, and/or If the methylation level of the DNA CpG site in the NNMT gene region is high, the tumor patient is suitable for prevention and/or treatment of mitochondrial oxidative phosphorylation pathway inhibitors.
  • the instructions or labels describe: when the mitochondrial oxidative phosphorylation pathway is down-regulated, the NNMT gene is overexpressed, the DNA methylase is under-expressed, the UHRF1 is under-expressed, and the NNMT gene nucleoside is down-regulated in the tumor cells of the tumor patient. If the methylation level of the acid site is low, and/or the methylation level of the DNA CpG site in the NNMT gene region is low, the tumor patient is not suitable for the prevention and/or treatment of mitochondrial oxidative phosphorylation pathway inhibitors.
  • a fifth aspect of the present invention provides a medicine box, the medicine box comprising:
  • NNMT gene expression level For detecting mitochondrial oxidative phosphorylation pathway expression level or activity, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene Reagents for the detection of methylation levels at CpG sites in DNA regions;
  • kit further includes instructions or labels.
  • the NNMT gene When the mitochondrial oxidative phosphorylation pathway is upregulated, the NNMT gene is underexpressed or not expressed, the DNA methylase is overexpressed, the UHRF1 is overexpressed, the NNMT gene nucleotide site methylation level is high, and/or The methylation level of DNA CpG sites in the NNMT gene region is high, and this tumor patient is suitable for prevention and/or treatment of mitochondrial oxidative phosphorylation pathway inhibitors.
  • the NNMT gene when the mitochondrial oxidative phosphorylation pathway is down-regulated, the NNMT gene is overexpressed, the DNA methylase is under-expressed, the UHRF1 is under-expressed, and the NNMT gene nucleotide site methylation level is low in the tumor cells of the tumor patient , and/or low methylation level of DNACpG sites in the NNMT gene region, the tumor patient is not suitable for prevention and/or treatment with mitochondrial oxidative phosphorylation pathway inhibitors.
  • the sixth aspect of the present invention provides a method for preventing and/or treating tumors, and administering an inhibitor of mitochondrial oxidative phosphorylation pathway to a subject in need thereof.
  • the tumor of the subject includes a tumor with low or no expression of NNMT gene.
  • the tumor of the subject includes a tumor with a high level of methylation at the DNA CpG site in the NNMT gene region.
  • the subject is human and non-human mammals (rodents, rabbits, monkeys, livestock, dogs, cats, etc.).
  • a seventh aspect of the present invention provides a device or system, the device or system comprising:
  • detection module described detection module is used to detect mitochondrial oxidative phosphorylation pathway expression level or activity, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methyl group methylation level, and/or methylation level of DNA CpG sites in the NNMT gene region;
  • an output module includes outputting the following information:
  • the NNMT gene When the mitochondrial oxidative phosphorylation pathway is upregulated, the NNMT gene is underexpressed or not expressed, the DNA methylase is overexpressed, the UHRF1 is overexpressed, the NNMT gene nucleotide site methylation level is high, and/or If the methylation level of the DNA CpG site in the NNMT gene region is high, the tumor patient is suitable for prevention and/or treatment with inhibitors of mitochondrial oxidative phosphorylation pathway; and/or
  • the NNMT gene When the mitochondrial oxidative phosphorylation pathway is down-regulated, the NNMT gene is over-expressed, the DNA methylase is under-expressed, the UHRF1 is under-expressed, the NNMT gene nucleotide site methylation level is low, and/or the NNMT gene region in tumor cells of tumor patients If the methylation level of DNA CpG sites is low, the tumor patients are not suitable for prevention and/or treatment with inhibitors of mitochondrial oxidative phosphorylation pathway.
  • the device includes a genetic detector or a protein detector.
  • the device or system further includes a sample injection module.
  • the sample injection module is used to inject tumor cell extracts.
  • the device or system further includes a data processing module.
  • the data processing module processes the obtained mitochondrial oxidative phosphorylation pathway expression level or activity level, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site High or low methylation level, and/or high or low methylation level of DNA CpG sites in the NNMT gene region.
  • the data processing module can obtain the expression level of NNMT gene and/or the methylation level of DNA CpG site in the promoter region of NNMT gene.
  • the data processing module obtains the level of NNMT gene expression and/or the methylation level of DNA CpG sites in the region from 1050bp before the NNMT gene transcription start site to 499bp after the gene transcription start site high and low.
  • the data processing module processes to obtain the level of NNMT gene expression and/or the methylation level of DNA CpG sites in the region from 1050 bp before the NNMT gene transcription start site to 193 bp before the gene transcription start site high and low.
  • the data processing module obtains the level of NNMT gene expression and/or the methylation level of DNA CpG sites in the region from 840 bp before the NNMT gene transcription start site to 469 bp before the gene transcription start site .
  • Figure 1 shows the inhibitory effect of different compounds on mitochondrial oxidative phosphorylation pathway (repeated 3 times in parallel).
  • Figure 2 shows the expression of ATF4 and p-s6 proteins in tumor cells after the mitochondrial oxidative phosphorylation pathway inhibitors Gboxin and Oligomycin A small molecules act on NCI-H82, G-401 and WSU-DLCL2 tumor cells.
  • Figure 3 shows the expression of ATF4 and p-s6 in tumor cells after the mitochondrial oxidative phosphorylation pathway inhibitors Gboxin and Oligomycin A small molecules act on SF126, CFPAC-1 and 786-O tumor cells.
  • Figure 4 shows the degree of cell-to-cell variation shown by the gene expression information of the cells.
  • Figure 5 shows functional differences between tumor cells sensitive and insensitive to inhibitors of the mitochondrial oxidative phosphorylation pathway.
  • Figure 6 shows the differences in metabolic pathways between tumor cells sensitive and insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • Figure 7 shows oxidative phosphorylation pathway protein complexes involved in expression.
  • Figure 8 shows mitochondrial membrane electrophoresis in cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, and WSU-DLCL2) and insensitive cell lines (786-O, CFPAC-1, and SF126). Difference.
  • FIG. 9 shows the mitochondrial oxygen consumption rate (OCR) of different tumor cells.
  • Figure 10 shows the genes with significant differences in expression screened out in different cells.
  • Figure 11 shows the correlation of mean NNMT gene transcription levels in tumor cells and tumor cells to inhibitors of the mitochondrial oxidative phosphorylation pathway.
  • Figure 12 shows the mRNA and protein expression of NNMT gene in different tumor cells, the upper figure shows the mRNA expression of NNMT gene, and the lower figure shows the protein expression of NNMT gene.
  • Figure 13 shows the expression of NNMT gene and methylation analysis of NNMT gene promoter region in different tumor cells.
  • Figure 14 shows the methylation levels of DNA CpG sites in the NNMT gene promoter region of tumor cells sensitive and insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • Figure 15 shows the methylation levels of DNA CpG sites in the region between 1050 bp before the transcription start site and 499 bp after the transcription start site of the NNMT gene in tumor cells sensitive and insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • Figure 16 shows the methylation levels of DNA CpG sites in the region between 1050 bp before the transcription start site and 193 bp before the transcription start site of the NNMT gene in tumor cells sensitive and insensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • Figure 17 shows the DNA CpG positions of 7 loci in the specific NNMT gene region of tumor cells sensitive and insensitive to mitochondrial oxidative phosphorylation pathway inhibitors, namely human chromosome 11 114165695, 114165730, 114165769, 114165804, 114165938, 114166050, 114166066, etc.
  • Spot methylation status black dots indicate that the relevant sites are methylated, white dots indicate that the relevant sites are not methylated, SST refers to the transcription start site, Chr11 refers to the human genome version according to GCF_000001405.25 (GRCh37.p13) Defining human chromosome 11.
  • Figure 18 shows adenosylmethionine (SAM) levels in tumor cells sensitive and insensitive to inhibitors of the mitochondrial oxidative phosphorylation pathway.
  • SAM adenosylmethionine
  • Figure 19 shows the correlation between the expression of NNMT and the expression of DNMT1, UHRF1, DNMT3a and DNMT3b in tumor cells.
  • Figure 20 shows the correlation of tumor cell DNMT1 gene transcription levels and tumor cells to mitochondrial oxidative phosphorylation pathway inhibitors.
  • Figure 21 shows the sensitivity of tumor cells to Gboxin after overexpressing NNMT protein in NCI-H82 cells by transgenic method and/or knocking down the expression of DNMT1 in NCI-H82 cells by transfecting shRNA, wherein Vector is normal expression NCI-H82 cells with NNMT protein and DNMT1; ov-NNMT is NCI-H82 cells overexpressed NNMT protein by transgene; sh-DNMT1#1 is NCI-H82 cells with knockdown of DNMT1 expression by sh-DNMT1#1, sh- DNMT1#2 is an NCI-H82 cell with knockdown of DNMT1 expression by sh-DNMT1#2, and ov-NNMT/sh-DNMT1#1 is an NCI that simultaneously overexpressed NNMT protein by transgene and knocked down DNMT1 expression by sh-DNMT1#1 -H82 cells; ov-NNMT/sh-DNMT1#2 are NCI-H82 cells that simultaneously overexpressed
  • Figure 22 shows the sensitivity of tumor cells to Oligomycin A after overexpressing NNMT protein in NCI-H82 cells by transgenic method and/or knocking down the expression of DNMT1 in NCI-H82 cells by transfecting shRNA, wherein Vector is normal NCI-H82 cells expressing NNMT protein and DNMT1; ov-NNMT is NCI-H82 cells overexpressing NNMT protein by transgenic; sh-DNMT1#1 is NCI-H82 cells knocking down DNMT1 expression by sh-DNMT1#1, sh - DNMT1#2 is NCI-H82 cells with knockdown of DNMT1 expression by sh-DNMT1#2, ov-NNMT/sh-DNMT1#1 is both transgenic to overexpress NNMT protein and knockdown of DNMT1 expression by sh-DNMT1#1 NCI-H82 cells; ov-NNMT/sh-DNMT1#2 are NCI-H82 cells that simultaneously overexpress NNMT protein by transgene
  • Figure 23 shows the NNMT protein content of NCI-H82 (ov-NNMT) overexpressing NNMT protein compared with normal NCI-H82 (Vector) detected by Western Blot experiment, wherein Vector is the NCI that normally expresses NNMT protein and DNMT1 -H82 cells; ov-NNMT is NCI-H82 cells transgenic to overexpress NNMT protein.
  • Figure 24 shows the detection of DNMT1 protein of NCI-H82 (sh-DNMT1#1 or sh-DNMT1#2) expressed by two shRNA knockdown tumor cells compared to normal NCI-H82 (shVector) by Western Blot assay shVector is the NCI-H82 cell that normally expresses DNMT1; shDNMT1#1 is the NCI-H82 cell that knocked down the expression of DNMT1 by sh-DNMT1#1, and shDNMT1#2 is the cell that knocked down the expression of DNMT1 by sh-DNMT1#2 NCI-H82 cells.
  • Figure 25 shows the inhibitory effect of oxidative phosphorylation pathway inhibitor S-Gboxin on NCI-H82 tumor-bearing cells, wherein NCI-H82 is a cell that normally expresses NNMT protein.
  • Figure 26 shows the inhibitory effect of oxidative phosphorylation pathway inhibitor S-Gboxin on NCI-H82-NNMT ov tumor bearing, wherein NCI-H82-NNMT ov is NCI-H82 cells overexpressing NNMT protein through transgenic.
  • Figure 27 shows the inhibitory effect of oxidative phosphorylation pathway inhibitor S-Gboxin on CFPAC-1 tumor bearing.
  • mitochondrial oxidative phosphorylation pathway inhibitors up-regulate mitochondrial oxidative phosphorylation pathway, low expression (or no expression) of NNMT gene, high expression of DNA methylase, and high expression of UHRF1 Tumor cells with high methylation levels at nucleotide sites of NNMT gene, and/or DNA CpG sites in the NNMT gene region have a significant inhibitory effect.
  • Mitochondrial oxidative phosphorylation pathway expression level or activity NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level and/or NNMT gene promoter DNA CpG site Methylation level can be used as a marker to judge whether tumor patients are suitable for prevention and/or treatment of mitochondrial oxidative phosphorylation pathway inhibitors. On this basis, the inventors have completed the present invention.
  • the terms “comprising,” “including,” and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”. As used herein, the terms “high level of DNA CpG site methylation”, “high level of DNA CpG site methylation” and “DNA CpG site hypermethylation” are used interchangeably.
  • low level of DNA CpG site methylation As used herein, the terms "low level of DNA CpG site methylation”, “low level of DNA CpG site methylation” and “DNA CpG site hypomethylation” are used interchangeably.
  • IC50 and “IC50” are used interchangeably and refer to the half-inhibiting concentration (50% inhibiting concentration), ie, the concentration of inhibitor at which 50% inhibitory effect is achieved.
  • CpG site methylation As used herein, the terms "CpG site methylation”, “CpG nucleotide methylation” and “CpG methylation” are used interchangeably.
  • Oligomycin A may be abbreviated as “Oligomycin”.
  • P/S refers to the addition of Penicillin and Streptomycin to the relevant medium.
  • a cell refers to a cell (eg, a single cancer cell) or a group of cells comprising a plurality of similar cells, etc. (eg, a tumor tissue).
  • a tumor patient suitable for use with an inhibitor of the mitochondrial oxidative phosphorylation pathway includes a tumor patient whose tumor is sensitive to an inhibitor of the mitochondrial oxidative phosphorylation pathway.
  • tumor patients are not candidates for mitochondrial oxidative phosphorylation pathway inhibitors
  • tumor patients are not candidates for mitochondrial oxidative phosphorylation pathway inhibitors
  • tumor patients are not sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • DNA CpG site methylation level refers to the expression level or activity of mitochondrial oxidative phosphorylation pathway, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level and One or more of the methylation levels of DNA CpG sites in the NNMT gene region.
  • up-regulated mitochondrial oxidative phosphorylation pathway low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high level of methylation at NNMT gene nucleotide sites, and/or NNMT gene
  • High methylation level of DNA CpG sites in the region refers to up-regulated mitochondrial oxidative phosphorylation pathway, low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, and methylation of NNMT gene nucleotide sites One or more of high levels and high levels of methylation at DNA CpG sites in the NNMT gene region.
  • Low methylation level refers to down-regulated mitochondrial oxidative phosphorylation pathway, high NNMT gene expression, low DNA methylase expression, low UHRF1 expression, low NNMT gene nucleotide site methylation level and NNMT gene region One or more of low methylation levels at DNA CpG sites.
  • NMT Nicotinamide N-Methyltransferase
  • base pair refers to base pair
  • SST refers to a transcription initiation site
  • Chr11 refers to human chromosome 11 as defined by the GCF_000001405.25 (GRCh37.p13) version of the human genome.
  • human chromosome 11 refers to human chromosome 11 as defined by the GCF_000001405.25 (GRCh37.p13) version of the human genome
  • the terms "before the transcription start site”, “after the transcription start site”, “before the transcription start site”, “after the transcription start site” do not include the transcription start site itself.
  • human chromosome 11 position 114165695" refers to the nucleotide position of human chromosome 11 position 114165695; "human chromosome 11 position 114165730” refers to the nucleotide position of human chromosome 11 position 114165730; "human chromosome 11 position 114165730" "Chromosomal No.
  • 114165769 refers to the nucleotide position of human chromosome 11 at position 114165769;
  • Human chromosome 11 position 114165804 refers to the nucleotide position of human chromosome 11 position 114165804;
  • Human chromosome 11 position 114165938 refers to human Nucleotide at position 114165938 of chromosome 11;
  • Human chromosome 11 at position 114166050 refers to the nucleotide at position 114166050 of human chromosome 11;
  • Human chromosome 11 at position 114166066 refers to the nucleotide at position 114166066 of human chromosome 11 acid.
  • S-adenosylmethionine is S-adenosyl methionine, abbreviated SAM.
  • gene expression includes the gene protein expression and/or the gene mRNA expression and the like.
  • DNMT3a refers to DNA methyltransferase 3a (DNA methyltransferase 3a).
  • DNMT3b refers to DNA methyltransferase 3b (DNA methyltransferase 3b).
  • DNMT1 refers to DNA methyltransferase 1 (DNA methyltransferase 1).
  • UHRF1 refers to ubiquitin-like PHD and RING finger domain-containing protein 1.
  • substituted refers to the replacement of a hydrogen atom on a group by a group other than a hydrogen atom, but which meets its valence requirements and results in a chemically stable compound, i.e., does not spontaneously undergo reactions such as cyclic Compounds that transform, eliminate, etc.
  • R1 As used herein, “R1” and “ R1” have the same meaning and are interchangeable with each other, and other similar definitions have the same meaning.
  • alkyl refers to a straight chain (ie, unbranched) or branched saturated hydrocarbon group containing only carbon atoms, or a combination of straight and branched chain groups.
  • a carbon number limitation such as C1-C6 alkyl
  • C1-C4 alkyl refers to an alkyl group containing 1-4 carbon atoms
  • Representative examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
  • halogen refers to F, Cl, Br or I.
  • halo refers to substitution with halogen.
  • haloalkyl means that one or more (preferably 1, 2, 3 or 4) hydrogens of an alkyl group are replaced by a halogen, wherein said alkyl and halogen are as defined above when preceding alkyl
  • a carbon number limitation (such as C1-C8 haloalkyl) means the alkyl group described contains 1-8 carbon atoms, for example, C1-C6 haloalkyl means a haloalkyl group containing 1-6 carbon atoms, representative examples include But not limited to -CF3, -CHF2, monofluoroisopropyl , difluorobutyl, or similar groups.
  • cycloalkyl refers to a group having a saturated or partially saturated monocyclic, bicyclic or polycyclic (fused, bridged or spiro) ring system.
  • a certain cycloalkyl group has a carbon number limitation (eg C3-C12), it means that the cycloalkyl group has 3-12 ring carbon atoms.
  • C3-C8 cycloalkyl refers to saturated or partially saturated monocyclic or bicycloalkyl groups having 3-8 ring carbon atoms, including cyclopropyl, cyclobutyl, cyclopentane , cycloheptyl, or similar groups.
  • halocycloalkyl means that one or more (preferably 1, 2, 3 or 4) hydrogens of a cycloalkyl group are replaced by a halogen, said cycloalkyl and halogen being as defined above,
  • a cycloalkyl group is preceded by a carbon number limitation (eg, C3-C8 haloalkyl)
  • C3-C8 haloalkyl means the cycloalkyl group in question contains 3-8 ring carbon atoms
  • C3-C8 haloalkyl means a halogen containing 3-6 carbon atoms
  • Cycloalkyl representative examples include, but are not limited to, monofluorocyclopropyl, monochlorocyclobutyl, monofluorocyclopentyl, difluorocycloheptyl, or the like.
  • alkoxy refers to a RO- group, wherein R is an alkyl group, and alkyl is as defined herein, when the alkoxy group is preceded by a carbon number limitation, such as C1-C8 alkoxy refers to the stated alkoxy group
  • the alkyl group in the group has 1-8 carbon atoms.
  • Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, or the like.
  • alkylthio refers to an RS- group in which R is an alkyl group and alkyl is as defined herein above with a carbon number limitation preceding the alkylthio group, as indicated by C1-C8 alkylthio groups
  • the alkyl group in the alkylthio group has 1 to 8 carbon atoms.
  • Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, tert-butylthio, or the like.
  • cycloalkoxy refers to an RO- group, wherein R is cycloalkyl, and cycloalkyl is as defined herein above with a carbon number limitation before cycloalkoxy, such as C3-C8 cycloalkoxy It means that the cycloalkyl group in the cycloalkoxy group has 3-8 carbon atoms.
  • Representative examples of cycloalkoxy include, but are not limited to, cyclopropoxy, cyclobutoxy, or the like.
  • cycloalkylthio refers to an RS- group wherein R is cycloalkyl, and cycloalkyl is as defined herein, with the number of carbon atoms before cycloalkylthio, such as C3-C8cycloalkylthio It means that the cycloalkyl group in the cycloalkylthio group has 3-8 carbon atoms.
  • Representative examples of cycloalkylthio include, but are not limited to, cyclopropylthio, cyclobutylthio, or the like.
  • haloalkoxy refers to a haloalkyl-O-, wherein the haloalkyl is as defined above, eg, C1-C6 haloalkoxy refers to a haloalkoxy containing 1-6 carbon atoms, representative examples Including, but not limited to, monofluoromethoxy, monofluoroethoxy, bisfluorobutoxy, or similar groups.
  • haloalkylthio refers to a haloalkyl-S- as defined above, eg, C1-C6 haloalkylthio refers to a haloalkylthio group containing 1-6 carbon atoms, representative examples Including, but not limited to, monofluoromethylthio, monofluoroethylthio, difluorobutylthio, or the like.
  • heterocycloalkyl refers to fully saturated or partially unsaturated cyclic groups (including, but not limited to, such as 3-7 membered monocyclic, 7-11 membered bicyclic, or 8-16 membered tricyclic systems) , in which at least one heteroatom is present in a ring with at least one carbon atom.
  • the number of members is limited before the heterocycloalkyl group, it refers to the number of ring atoms of the heterocycloalkyl group, for example, a 3-16-membered heterocycloalkyl group refers to a heterocycloalkyl group having 3-16 ring atoms.
  • Each heterocyclic ring containing heteroatoms may carry one or more (eg 1, 2, 3 or 4) heteroatoms, each of which is independently selected from nitrogen, oxygen or sulfur, wherein nitrogen Or the sulfur atom can be oxidized and the nitrogen atom can also be quaternized.
  • a heterocycloalkyl group can be attached to the residue of any heteroatom or carbon atom of the ring or ring system molecule.
  • Typical monocyclic heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 4-piperidinyl, tetrahydropyranyl, and the like .
  • Polycyclic heterocycloalkyl groups include spiro, fused and bridged heterocyclic groups; the spiro, fused and bridged heterocycloalkyl groups are optionally connected to other groups through a single bond, or It is further connected to other cycloalkane rings and heterocycles through any two or more atoms on the ring.
  • aryl refers to an all-carbon monocyclic or fused polycyclic (that is, rings that share adjacent pairs of carbon atoms) groups with a conjugated ⁇ -electron system, and is an aromatic cyclic hydrocarbon compound group, when An aryl group preceded by a carbon number limitation, such as a C6-C12 aryl group, means that the aryl group has 6-12 ring carbon atoms, such as phenyl and naphthyl.
  • arylene refers to a group formed by the loss of one hydrogen atom from an aryl group, the aryl group is as defined above, and when the arylene group is preceded by a carbon number limitation, such as a C6-C12 arylene group, it refers to the described aryl group
  • Arylene groups have 6-12 ring carbon atoms, and representative examples of arylene groups include, but are not limited to, phenylene, naphthylene, and the like.
  • heteroaryl refers to an aromatic heterocyclic ring system group having one to more (preferably 1, 2, 3 or 4) heteroatoms, which may be monocyclic (monocyclic) or fused together Or covalently linked polycyclic rings (bicyclic, tricyclic or polycyclic), each heterocyclic ring containing a heteroatom may have one or more (eg 1, 2, 3, 4) each independently A heteroatom selected from the group consisting of oxygen, sulfur and nitrogen.
  • a 5-12-membered heteroaryl group refers to a heteroaryl group with 5-12 ring atoms, a representative example Including but not limited to: pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl azolyl and tetrazolyl.
  • heteroarylene refers to a group formed by the loss of a hydrogen atom from a heteroaryl group, the heteroaryl group is as defined above, when the heteroarylene group is preceded by a carbon number limitation, such as a C6-C12 heteroarylene group, It means that the heteroarylene group has 6-12 ring carbon atoms, and representative examples of the heteroarylene group include but are not limited to pyrrolidene, pyrazolylide, imidazolylide, triazolylidene, oxadiazolyl and oxazolylide and the like.
  • hydrogen atoms are replaced by substituents selected from the group consisting of C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl (such as trifluoromethyl), C3- C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio , C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10-membered heteroaryl, methanesulfonyl, sulfonyl.
  • an optionally substituted group may have at any substitutable position of the group a substituent selected from a specified group, which may be the same or different at each position.
  • prevention refers to a method of preventing the onset of a disease and/or its attendant symptoms or protecting a subject from acquiring a disease.
  • Treatment includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • the inhibitor of the mitochondrial oxidative phosphorylation pathway of the present invention reduces the associated disease (eg, tumor) and its concomitant diseases compared to the levels observed in the absence of the inhibitor of the mitochondrial oxidative phosphorylation pathway of the present invention Symptoms are reduced, inhibited and/or reversed, eg, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, or 100%.
  • Oxidative Phosphorylation is one of the most important pathways in mitochondria, which utilizes NADH and FADH derived from pathways such as the tricarboxylic acid cycle and fat oxidation to synthesize ATP.
  • Mitochondrial oxidative phosphorylation pathway consists of more than 90 proteins, these proteins form five protein complexes, complexes I, II, III, IV and V.
  • the first 4 protein complexes also known as the electron transport chain, receive electrons from electron donors NADH and FADH and transfer them to oxygen.
  • mitochondrial oxidative phosphorylation pathway is very important for cell growth and is related to many diseases, such as cancer, immune-related diseases, neurodegenerative diseases, and inhibition of mitochondrial oxidative phosphorylation pathway can be used to treat cancer, immune-related diseases, neurodegenerative diseases Diseases, especially cancer cells with stem cell properties with a high degree of malignancy, are extremely dependent on this pathway for survival. Inhibiting this pathway can effectively kill such cancer cells, thereby solving the problem of related malignant cancer recurrence.
  • NNMT Nicotinamide N-Methyltransferase
  • different databases have different identification numbers for the NNMT gene: HGNC:7861; Entrez Gene:4837; Ensembl:ENSG00000166741; OMIM:600008; UniProtKB:P40261.
  • the NNMT gene region is located in the 114,128,528 bp to 114,184,258 bp of human chromosome 11, with a total DNA sequence of 55,731 bp, including the NNMT gene promoter region and the NNMT gene exon NNMT gene intron region and the NNMT gene transcription start site is 114,166,535 bp.
  • the NNMT gene promoter region is the nucleotide sequence from 114,164,535 bp to 114,167,034 bp of human chromosome 11, that is, 2000bp before the transcription start site of the NNMT gene (bold part) to the transcription start site itself and 499bp after it ( The sequence between the underlined parts), the region with a total length of 2500bp is the NNMT gene promoter region, and the nucleotide sequence of the NNMT gene promoter region is shown in the following SEQ ID NO: 1:
  • 1050bp before the transcription initiation site of the NNMT gene to 499bp after the transcription initiation site of the gene is the 951-2500 position of the nucleotide sequence shown in SEQ ID NO: 1.
  • 1050bp before the transcription initiation site of the NNMT gene to 193bp before the transcription initiation site of the gene are positions 951-1808 of the nucleotide sequence shown in SEQ ID NO:1.
  • the 840 bp before the transcription initiation site of the NNMT gene to the 469 bp before the transcription initiation site of the gene are positions 1161-1532 of the nucleotide sequence shown in SEQ ID NO: 1.
  • the sites of human chromosome 11 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 correspond to the sites of the nucleotide sequence of SEQ ID NO: 1 as shown in Table 1 below:
  • the locus of human chromosome 11 The position corresponding to the nucleotide sequence of SEQ ID NO: 1 114165695 bits 1161st 114165730 bits No. 1196 114165769 bits No. 1235 114165804 bits No. 1270 114165938 bits No. 1404 114166050 bits No. 1516 114166066 bits No. 1532
  • DNA methylation is a form of chemical modification of DNA, which can change the genetic performance without changing the DNA sequence.
  • a large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability and the way DNA interacts with proteins, thereby regulating gene expression.
  • DNA methylation is one of the earliest discovered and most deeply studied epigenetic regulatory mechanisms.
  • DNA methylation refers to the conversion of specific bases on the DNA sequence to S-adenosyl methionine (SAM) under the catalysis of DNA methyltransferase (DNMT).
  • SAM S-adenosyl methionine
  • DNMT DNA methyltransferase
  • DNA methylation involved in general research mainly refers to the methylation process that occurs at the 5th carbon atom on cytosine in CpG dinucleotides, and the product is called 5-methylcytosine (5-mC) , is the main form of DNA methylation in plants, animals and other eukaryotes.
  • DNA methylation as a relatively stable modification state, under the action of DNA methyltransferase, can be inherited to the new offspring DNA along with the DNA replication process, which is an important epigenetic mechanism.
  • DNA methylation reactions are divided into 2 types. One is that both strands of unmethylated DNA are methylated, which is called de novo methylation; the other is that one strand of double-stranded DNA is already methylated and the other is unmethylated Methylated chains are methylated, a type called maintenance methylation.
  • DNA methylation is DNA CpG site methylation.
  • the distribution of CpG dinucleotides in the human genome is very heterogeneous, and in certain segments of the genome, CpGs remain at or above normal probability.
  • CpG site-enriched regions also known as CpG islands
  • CpG island are mainly located in the promoter and exon regions of genes, and are some regions rich in CpG dinucleotides.
  • About 60% of gene promoters contain CpG island.
  • CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G).
  • DNA methylation modification is an important way for epigenetic modification to regulate gene expression, and the level of DNA methylation in a specific gene region often affects the expression level of the gene. Compared with the regulation of gene expression by signal transduction pathways and transcription factors, DNA methylation modification in epigenetic modification has a more stable effect on gene expression, and is not easily affected by the extracellular environment. DNA methylation modification is easy to use. The existing technology can accurately detect it, so it is an ideal biomarker.
  • the present invention provides a mitochondrial oxidative phosphorylation pathway inhibitor for preventing and/or treating tumors (cancer).
  • the inhibitor of mitochondrial oxidative phosphorylation pathway comprises compounds of formula I, formula II and/or formula III, or optical isomers or racemates thereof, or solvates thereof, or a pharmaceutically acceptable salt thereof.
  • a compound of formula I of the present invention or “a compound of formula I” are used interchangeably and refer to a compound of formula I, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable compound thereof. acceptable salt. It should be understood that the term also includes mixtures of the above components.
  • a compound of formula II of the present invention or “a compound of formula II” are used interchangeably and refer to a compound of formula II, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable compound thereof. acceptable salt. It should be understood that the term also includes mixtures of the above components.
  • a compound of formula III of the present invention or “a compound of formula III” are used interchangeably and refer to a compound of formula III, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable compound thereof. acceptable salt. It should be understood that the term also includes mixtures of the above components.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention with an acid or base suitable for use as a medicament.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are those formed by the compounds of the present invention with acids.
  • Suitable salt-forming acids include, but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydrofluoric acid, hydroiodic acid, sulfuric acid, nitric acid, Inorganic acids such as phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid , organic acids such as benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • a class of preferred salts is the metal salts formed by the compounds of the present invention and bases.
  • the bases suitable for forming salts include (but are not limited to): inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, Ammonia, triethylamine, diethylamine and other organic bases.
  • the compound represented by the formula I of the present invention can be converted into its pharmaceutically acceptable salt by conventional methods.
  • the solution of the corresponding acid can be added to the solution of the above compound, and the solvent can be removed after the salt is completely formed.
  • the mitochondrial oxidative phosphorylation pathway inhibitor is selected from the group consisting of:
  • the research of the present invention shows that the compound of the present invention can up-regulate mitochondrial oxidative phosphorylation pathway, low expression (or no expression) of NNMT gene, high expression of DNA methylase, high expression of UHRF1, and methylation level of NNMT gene nucleotide site.
  • Tumor cells with high and/or high methylation levels of DNA CpG sites in the NNMT gene region have more significant inhibitory effects, up-regulated mitochondrial oxidative phosphorylation pathway, low expression (or no expression) of NNMT gene, and high DNA methylase Tumor cells with high expression, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or high methylation level of DNA CpG site in NNMT gene region are sensitive to the mitochondrial oxidative phosphorylation pathway inhibitor of the present invention.
  • the research of the present invention shows that the mitochondrial oxidative phosphorylation pathway inhibitor of the present invention can be used to prevent and/or treat tumors.
  • the tumor described in the present invention includes a tumor with up-regulated mitochondrial oxidative phosphorylation pathway.
  • the tumor with up-regulated mitochondrial oxidative phosphorylation pathway of the present invention is as described above in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with low or no expression of NNMT gene.
  • tumors with low expression or no expression of the NNMT gene of the present invention are as described above in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with high DNA methylase expression.
  • the tumor with high DNA methylase expression of the present invention is as described in the first aspect of the present invention.
  • the DNA methylases described in the present invention include, but are not limited to, DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the DNA methylase of the present invention includes DNMT1.
  • the tumor described in the present invention includes a tumor with high expression of DNMT1.
  • the tumors with high DNMT1 expression of the present invention are as described in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with high expression of DNMT3a.
  • the tumors with high DNMT3a expression of the present invention are as described in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with high expression of DNMT3b.
  • the tumors with high DNMT3b expression of the present invention are as described in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with high expression of UHRF1 (ubiquitin-like PHD and RING finger domain protein 1).
  • UHRF1 ubiquitin-like PHD and RING finger domain protein 1
  • the tumors with high UHRF1 expression of the present invention are as described in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with a high level of methylation at the nucleotide site of the NNMT gene.
  • the tumor with high methylation level of the NNMT gene nucleotide site of the present invention is as described in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with a high level of methylation at the DNA CpG site in the NNMT gene region.
  • tumors with high methylation levels of DNA CpG sites in the NNMT gene region of the present invention are as described in the first aspect of the present invention.
  • the tumor of the present invention is as described above in the first aspect of the present invention.
  • tumor types corresponding to representative tumor cell lines are shown in Table 2 below:
  • tumor cell line Corresponding tumor type NCI-H82 human small cell lung cancer cells G-401 Human kidney cancer Wilms cells MDA-MB-453 breast cancer cells WSU-DLCL2 Human diffuse large B lymphoma cells SU-DHL-2 large cell lymphoma cells OCI-AML-3 FAB M4 grade AML acute myeloid leukemia SW48 human colon adenocarcinoma cells ATN-1 T cell leukemia cells HCC15 non-small cell lung cancer cells OCI-LY-19 B cell lymphoma cells
  • 22RV1 prostate cancer cells MIA PaCa-2 Pancreatic cancer cells CCRF-CEM acute T lymphoblastic leukemia cells HH skin T cell lymphoma cells OCI-AML-5 M4 grade AML acute myeloid leukemia cells G-402 kidney cancer cells HCC1806 breast cancer cells BT-549 breast cancer cells OCI-AML-4 acute myeloid leukemia cells H9 lymphoma cells Jurkat,Clone E6-1 T lymphoma cells G-361 melanoma cells U-937 histiocytic lymphoma cells SNU-398 hepatocellular carcinoma NCI-H1048 small cell lung cancer cells A-375 melanoma cells D283 Med Medulloblastoma cells GAK melanoma cells CHL-1 melanoma cells NCI-H1155 non-small cell lung cancer cells LS 180 colorectal adenocarcinoma cells Daoy Medulloblastoma cells DU 145 brain metastases prostate cancer cells AM
  • kidney cancer cells COLO 320HSR colorectal adenocarcinoma cells
  • CFPAC-1 Pancreatic cancer cells
  • SF126 brain tumor cells 786-O renal clear cell adenocarcinoma
  • the present invention also provides a marker for judging whether a tumor patient is suitable for prevention and/or treatment with an inhibitor of mitochondrial oxidative phosphorylation pathway, the marker includes the expression level or activity of mitochondrial oxidative phosphorylation pathway, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level.
  • mitochondrial oxidative phosphorylation pathway expression level or activity NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene
  • the methylation level of DNA CpG sites in the region is used as a marker to determine whether tumor patients are suitable for prevention and/or treatment with mitochondrial oxidative phosphorylation pathway inhibitors, and the methods include but are not limited to:
  • the NNMT gene When the mitochondrial oxidative phosphorylation pathway is upregulated, the NNMT gene is underexpressed or not expressed, the DNA methylase is overexpressed, the UHRF1 is overexpressed, the NNMT gene nucleotide site methylation level is high, and/or If the methylation level of the DNA CpG site in the NNMT gene region is high, the tumor patient is suitable for prevention and/or treatment with inhibitors of mitochondrial oxidative phosphorylation pathway; and/or
  • the NNMT gene When the mitochondrial oxidative phosphorylation pathway is down-regulated, the NNMT gene is over-expressed, the DNA methylase is under-expressed, the UHRF1 is under-expressed, the NNMT gene nucleotide site methylation level is low, and/or the NNMT gene region in tumor cells of tumor patients If the methylation level of DNA CpG sites is low, the tumor patients are not suitable for prevention and/or treatment with inhibitors of mitochondrial oxidative phosphorylation pathway.
  • tumors with up-regulated mitochondrial oxidative phosphorylation pathway tumors with low or no expression of NNMT gene, tumors with high expression of DNA methylase (such as DNMT1), tumors with high expression of UHRF1, tumors with high expression of NNMT gene, Tumors with high levels of acid site methylation,
  • tumors with down-regulated mitochondrial oxidative phosphorylation pathway tumors with high NNMT gene expression, tumors with low DNA methylase (such as DNMT1) expression, tumors with low UHRF1 expression, and NNMT gene nucleotide sites described in the present invention
  • Tumors with low methylation levels, and/or tumors with low methylation levels of DNA CpG sites in the NNMT gene region are as described above in the second aspect of the present invention.
  • compositions or formulations, combinations of active ingredients and kits and methods of administration are provided.
  • composition of the present invention is preferably a pharmaceutical composition, and the composition of the present invention may include a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid, semi-solid, liquid or gel fillers which are suitable for human or animal use and which must be of sufficient purity and low enough toxicity. "Compatibility” refers to the components in the pharmaceutical composition and the active ingredients of the drug and their intermingling with each other without significantly reducing the efficacy of the drug.
  • the pharmaceutically acceptable carrier is not particularly limited, and materials commonly used in the art can be selected, or prepared by conventional methods, or purchased from the market.
  • pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, skin penetration enhancers, colorants, flavors, stabilizers, antioxidants, preservatives ,
  • cellulose and its derivatives such as
  • the dosage form of the composition or preparation is a solid preparation, a liquid preparation or a semi-solid preparation.
  • the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation
  • the dosage form of the composition or preparation is tablet, injection, infusion, ointment, gel, solution, microsphere or film.
  • the drug formulation should match the mode of administration.
  • the agents of the present invention may also be used (including before, during or after use) with other synergistic therapeutic agents.
  • a safe and effective amount of the drug is administered to the desired subject (eg, a human or non-human mammal), said safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases Not to exceed about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the invention provides biomarkers for mitochondrial oxidative phosphorylation pathway inhibitor drugs that can guide their precise drug use, and the relevant biomarkers can effectively identify tumor patients who are sensitive to such antitumor drugs, improve their therapeutic effects, and avoid the need for such drugs. Such drugs are administered to patients with tumors that are not sensitive to them, so that the precise application of such drugs can be realized.
  • the present invention is the first to unexpectedly find through systematic research the expression level or activity of mitochondrial oxidative phosphorylation pathway, NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or
  • the methylation level of DNA CpG sites in the NNMT gene region can be used as a marker to determine whether specific tumor cells are suitable for treatment with mitochondrial oxidative phosphorylation pathway inhibitors.
  • Tumors with high levels of methylation are highly sensitive to mitochondrial oxidative phosphorylation pathway inhibitor drugs, that is, oxidative phosphorylation pathway inhibitor drugs upregulate mitochondrial oxidative phosphorylation pathway, low or no expression of NNMT gene, and high expression of DNA methylase.
  • Tumors with high expression of UHRF1, high nucleotide methylation level of NNMT gene and/or high methylation level of DNA CpG site in NNMT gene region have excellent therapeutic effect.
  • the detection method of DNA CpG site methylation level is mature, stable and reliable, which is suitable for the development of molecular markers.
  • Oligomycin A, Gboxin, S-Gboxin, and IACS-010759 are all known inhibitors of mitochondrial oxidative phosphorylation pathway.
  • the structural formula of the IACS-010759 compound is as follows:
  • DNMT3a refers to DNA methyltransferase 3a, NCBI entrez gene: 1788; Uniprotkb/Swiss-port: Q9Y6K1.
  • DNMT3b refers to DNA methyltransferase 3b, NCBI entrez gene: 1789; Uniprotkb/Swiss-port: Q9UBC3.
  • DNMT1 refers to DNA methyltransferase 1, NCBI entrez gene: 1786; Uniprotkb/Swiss-port: P26358.
  • UHRF1 refers to ubiquitin-like PHD and RING finger domain-containing protein 1, NCBI entrez gene: 29128; Uniprotkb/Swiss-port: Q96T88.
  • NNMT gene is Nicotinamide N-Methyltransferase.
  • the nucleotide sequence of the NNMT gene promoter region is shown in SEQ ID NO: 1.
  • 1050bp before the transcription initiation site of the NNMT gene to 499bp after the transcription initiation site are positions 951-2500 of the nucleotide sequence shown in SEQ ID NO: 1.
  • 1050bp before the transcription start site of the NNMT gene to 193bp before the transcription start site are positions 951-1808 of the nucleotide sequence shown in SEQ ID NO: 1.
  • 840bp before the transcription start site of NNMT gene to 469bp before the transcription start site are positions 1161-1532 of the nucleotide sequence shown in SEQ ID NO: 1.
  • Oxygen required by cells is mainly consumed by mitochondrial oxidative phosphorylation pathway.
  • the analysis of mitochondrial oxygen consumption rate (OCR) can directly reflect the activity of mitochondrial oxidative phosphorylation pathway.
  • OCR mitochondrial oxygen consumption rate
  • the Seahorse XFe metabolism analyzer was used to detect the oxygen consumption of NCI-H82 cells in the presence and absence of mitochondrial oxidative phosphorylation pathway inhibitors Oligomycin A, Gboxin, S-Gboxin and IACS-010759.
  • NCI-H82 (derived from ATCC, code HTB-175) cells were cultured in RPMI1640 medium supplemented with 10% FBS (plus P/S), and the mitochondrial oxidative phosphorylation pathway inhibitor Oligomycin A (1 ⁇ M) was added respectively.
  • Oligomycin A (1 ⁇ M) was added respectively.
  • Gboxin (2 ⁇ M), S-Gboxin (5 ⁇ M), IACS-010759 (1 ⁇ M), and a control group without any mitochondrial oxidative phosphorylation pathway inhibitor the detection of cellular oxygen consumption was completed within 0.5 hours. The results are shown in Figure 1.
  • Cell lines with different genotypes were randomly selected from different tissues, and their sensitivity to mitochondrial oxidative phosphorylation pathway inhibitor Gboxin was detected by using cell viability detection reagent. While some cell lines were not sensitive to Gboxin compounds, IC 50 values were higher.
  • Cell line NCI-H82 (ATCC, No. HTB-175) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line G-401 (ATCC, No. CRL-1441) was cultured in McCoy's 5a medium + P/S containing 10% fetal bovine serum;
  • Cell line MDA-MB-453 (ATCC, No. HTB-131) was cultured in Leibovitz's L-15 medium + P/S containing 10% fetal bovine serum;
  • the cell line WSU-DLCL2 (DSMZ, No. ACC-575) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line SU-DHL-2 (ATCC, No. CRL-2956) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • the cell line OCI-AML-3 (DSMZ, No. ACC-582) was cultured in RPMI1640 medium + P/S containing 20% fetal bovine serum;
  • Cell line SW48 (ATCC, number CCL-231) was cultured in Leibovitz's L-15 medium + P/S containing 10% fetal bovine serum;
  • Cell line ATN-1 (RIKEN, No. RBRC-RCB1440) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum and 0.1 mM NEAA;
  • Cell line HCC15 (KCLB, No. 70015) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • the cell line OCI-LY-19 (DSMZ, code ACC-528) was cultured in 80-90% alpha-MEM medium + P/S containing 10-20% h.i.FBS;
  • Cell line 22RV1 (ATCC, No. CRL-2505) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line MIA PaCa-2 (ATCC, No. CRL-1420) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line CCRF-CEM (ATCC, No. CCL-119) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line HH (ATCC, No. CRL-2105) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • the cell line OCI-AML-5 (DSMZ, No. ACC-247) was cultured in alpha-MEM medium (medium adjusted by 5637 cell line containing 20% fetal bovine serum and 10% volume fraction) + P/S;
  • Cell line G-402 (ATCC, number CRL-1440) was cultured in McCoy's 5a medium + P/S containing 10% fetal bovine serum;
  • Cell line HCC1806 (ATCC, No. CRL-2335) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line BT-549 (ATCC, No. HTB-122) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum 0.023IU/ml human insulin;
  • the cell line OCI-AML-4 (DSMZ, No. ACC-729) was cultured in alpha-MEM medium (medium adjusted by 5637 cell line containing 20% fetal bovine serum and 20% volume fraction) + P/S;
  • Cell line H9 (ATCC, No. HTB-176) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line Jurkat, Clone E6-1 (ATCC, No. TIB-152) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line G-361 (ATCC, accession CRL-1424) was cultured in McCoy's 5a medium + P/S with 10% fetal bovine serum.
  • Cell line U-937 (ATCC, No. CRL-1593.2) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line SNU-398 (ATCC, No. CRL-2233) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • the cell line NCI-H1048 (ATCC, No. CRL-5853) was cultured in HITES medium + P/S containing 5% fetal bovine serum;
  • Cell line A-375 (ATCC, No. CRL-1619) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line D283 Med (ATCC, No. HTB-185) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line GAK (JCRB, No. JCRB0180) was cultured in Ham's F12 medium + P/S containing 20% fetal bovine serum;
  • Cell line CHL-1 (ATCC, No. CRL-9446) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line NCI-H1155 (ATCC, No. CRL-5818) was cultured in serum-free ACL-4 medium + P/S;
  • Cell line LS 180 (ATCC, No. CL-187) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line Daoy (ATCC, No. HTB-186) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line DU 145 (ATCC, No. HTB-81) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line AM-38 (JCRB, No. IFO50492) was cultured in EMEM medium + P/S containing 20% heat-inactivated fetal bovine serum;
  • Cell line HCC70 (ATCC, No. CRL-2315) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line PANC-1 (ATCC, No. CRL-1469) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line U-87 MG (ATCC, number HTB-14) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line MJ (ATCC, No. CRL-8294) was cultured in IMDM medium + P/S containing 20% fetal bovine serum;
  • Cell line Gp2D (ECACC, No. 95090714) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line SU.86.86 (ATCC, No. CRL-1837) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line NCI-H2081 (ATCC, No. CRL-5920) was cultured in HITES medium + P/S containing 5% fetal bovine serum;
  • Cell line NCI-H1793 (ATCC, No. CRL-5896) was cultured in HITES medium + P/S containing 5% fetal bovine serum;
  • Cell line ACHN (ATCC, No. CRL-1611) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • the cell line U-251 MG (ECACC, No. 9063001) was cultured in EMEM medium + P/S containing 2mM Glutamine, 1% NEAA, 1 mM Sodium Pyruvate (NaP) and 10% fetal bovine serum;
  • Cell line MDA-MB-231 (ATCC, No. HTB-26) was cultured in Leibovitz's L-15 medium + P/S containing 10% fetal bovine serum;
  • Cell line NCI-H196 (ATCC, No. CRL-5823) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line PC-3 (ATCC, No. CRL-1435) was cultured in F-12K medium + P/S containing 10% fetal bovine serum;
  • the cell line OCI-M1 (DSMZ, No. ACC-529) was cultured in IMDM medium + P/S containing 20% fetal bovine serum;
  • the cell line NCI-H1651 (ATCC, No. CRL-5884) was cultured in ACL-4 medium + P/S containing 10% fetal bovine serum;
  • Cell line C3A (ATCC, No. CRL-10741) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line SNU-449 (ATCC, No. CRL-2234) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line GB-1 (JCRB, No. IFO50489) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line 769-P (ATCC, No. CRL-1933) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line COLO 320HSR (ATCC, number CCL-220.1) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line CFPAC-1 (ATCC, No. CRL-1918) was cultured in IMDM medium + P/S containing 10% fetal bovine serum;
  • Cell line SF126 (JCRB, No. IFO50286) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • Cell line 786-O (ATCC, No. CRL-1932) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • IC 50 is the half-inhibiting concentration (50% inhibiting concentration), that is, the concentration of the inhibitor when the 50% inhibitory effect is achieved.
  • the tumor cell lines (NCI-H82, G-401, MDA-MB-453, WSU-DLCL2, SW48) that are sensitive to the mitochondrial oxidative phosphorylation pathway inhibitor Gboxin small molecule found in Example 2 were further tested using cell viability detection reagents
  • the sensitivity of and insensitive tumor cell lines (GB-1, CFPAC-1, SF126, 786-O) to mitochondrial oxidative phosphorylation pathway inhibitors Oligomycin A and IACS-010759.
  • IC 50 is the half-inhibiting concentration (50% inhibiting concentration), that is, the concentration of the inhibitor when 50% inhibitory effect is achieved.
  • NCI-H82, G-401 and WSU-DLCL2 tumor cells were cultured in the relevant medium containing 10% FBS (plus p/s), and after the cells were passaged overnight, 1 ⁇ M and 3 ⁇ M were added as shown in Figure 2.
  • the Gboxin and 1 ⁇ M Oligomycin A were treated for 12 hours to detect the protein content of ATF4 and p-S6 protein by Western Blot experiment. The experimental results are shown in Figure 2.
  • the ATF4 stress pathway is up-regulated when the cell lines NCI-H82, G-401 and WSU-DLCL2 are acted on by small molecules such as Gboxin and Oligomycin A, which are inhibitors of the mitochondrial oxidative phosphorylation pathway, while the decrease in p-S6 protein indicates that mTOR Pathway activity was inhibited, indicating that the oxidative phosphorylation pathway was active in these cells and sensitive to oxidative phosphorylation pathway inhibitors.
  • Gboxin and Oligomycin A are inhibitors of the mitochondrial oxidative phosphorylation pathway
  • Sensitive cell lines NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2
  • insensitive cell lines (786-O , CFPAC-1, GB-1, SF126) gene transcription and expression.
  • the behavior and characteristics of cells are determined by the genes expressed by the cells. Through whole-genome gene transcription sequencing, the mRNA transcription levels of each gene in a certain cell can be accurately obtained, and the transcription levels of all gene mRNAs can be analyzed by bioinformatics calculation. Different cells can be classified according to the similarity of gene expression.
  • Bioinformatics analysis of cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive cell lines (786-O , CFPAC-1, GB-1, SF126) the function of differentially expressed genes.
  • cells sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive cells (786-O, CFPAC-1 , GB-1, SF126) differentially expressed up-regulated genes are mainly concentrated in metabolism-related pathways (such as carbon metabolism, pyruvate metabolism, propionate metabolism, glyoxylate and dicarboxylic acid metabolism, etc.), indicating that these two There were significant differences in cellular metabolism among population cells, and related metabolic pathways were up-regulated in sensitive cells.
  • metabolism-related pathways such as carbon metabolism, pyruvate metabolism, propionate metabolism, glyoxylate and dicarboxylic acid metabolism, etc.
  • Bioinformatics analysis of cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive cell lines (786-O , CFPAC-1, GB-1, SF126) major differences in metabolic pathways.
  • Bioinformatics analysis of tumor cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive tumor cell lines (786 -O, CFPAC-1, GB-1, SF126) in oxidative phosphorylation pathway protein complexes.
  • Example 8 cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive cell lines (786 -O, CFPAC-1, GB-1, SF126) had significant differences in mitochondrial oxidative phosphorylation pathway, which was composed of 5 protein complexes containing more than 90 proteins.
  • the cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive cell lines (786-O, CFPAC -1, GB-1, SF126) differentially expressed genes in the oxidative phosphorylation pathway were mainly concentrated in the oxidative phosphorylation pathway protein complexes I, III, IV, and V, which were highly expressed in sensitive cells.
  • the mitochondrial membrane electrical difference can regulate mitochondrial protein transport, autophagy, ATP synthesis and other important mitochondrial functions.
  • Cells sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401 and WSU-DLCL2) and Insensitive cells (786-O, CFPAC-1, and SF126) showed significant differences in oxidative phosphorylation pathways, and these differences were also reflected in mitochondrial membrane potential differences.
  • the mitochondrial membrane differential indicator TMRE tetramethylrhodamine, ethyl ester was used to detect cells that were sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401 and WSU-DLCL2) and those that were not in normal culture.
  • the membrane potential of cells sensitive to mitochondrial oxidative phosphorylation pathway inhibitors was relatively high, and the membrane potential of insensitive cells (786-O, CFPAC-1 and SF126) was relatively high.
  • mitochondrial membrane potential was relatively low, and there was a significant difference in mitochondrial membrane potential between the two groups of cells.
  • Biomarkers provide an excellent technical guarantee for the realization of precision medicine.
  • a good biomarker should have the ability to clearly distinguish between drug responders and non-responders.
  • a biomarker is the expression of a gene or group of genes.
  • cells sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and insensitive cells (786-O, CFPAC-1, GB-1, SF126) had very large differences in NNMT gene expression, and NNMT gene expression was low in oxidative phosphorylation pathway inhibitor-sensitive cells.
  • Biomarkers provide an excellent technical guarantee for the realization of precision medicine.
  • a good biomarker should have the ability to clearly distinguish between drug responders and non-responders, and be proportional or inversely proportional to the corresponding biological characteristics.
  • the NNMT gene transcription level of each cell is negatively correlated with its sensitivity to Gboxin (the smaller the IC 50 , the more sensitive it is), indicating that the NNMT gene transcription level and tumor cells are sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the sensitivity of the tumor cells was negatively correlated, that is, the lower the NNMT gene transcription level, the higher the sensitivity of the tumor cells to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the NNMT gene was validated in terms of mRNA and protein expression levels in cells sensitive and insensitive to inhibitors of the mitochondrial oxidative phosphorylation pathway.
  • the mRNA and protein of NNMT gene were lowly expressed in cells sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, SW48 and WSU-DLCL2), while It is highly expressed in cells insensitive to mitochondrial oxidative phosphorylation pathway inhibitors (786-O, CFPAC-1 and SF126).
  • the experimental results are shown in Figure 13.
  • the X-axis in the figure represents the ratio of the transcriptional expression level of a certain gene in sensitive cells and insensitive cells (sensitive cells/insensitive cells), and the Y-axis represents the CpG island in the promoter region of a gene.
  • the NNMT gene promoter region is hypermethylated in cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) It was hypomethylated and highly expressed in insensitive tumor cell lines (786-O, CFPAC-1, GB-1 and SF126).
  • NCI-H82 Five tumor cell lines (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) that were sensitive to mitochondrial oxidative phosphorylation pathway inhibitors such as Oligomycin A and Gboxin and four tumor cell lines that were insensitive (786-O, CFPAC-1, GB-1 and SF126) NNMT gene promoter region, the region between 1050bp before the transcription start site of NNMT gene and 499bp after the transcription start site, and the transcription start site of NNMT gene The region between the first 1050bp and the first 193bp of the transcription start site was subjected to bisulfite sequencing to detect the methylation level of DNA CpG sites in the relevant region.
  • Figure 14 promoter region of NNMT gene
  • Figure 15 region between 1050bp before transcription start site of NNMT gene and 499bp after transcription start site
  • Figure 16 (1050bp before transcription start site of NNMT gene to transcription start site)
  • mitochondrial oxidative phosphorylation pathway inhibitor on the promoter region of NNMT gene the region between 1050bp before the transcription start site of NNMT gene and 499bp after the transcription start site
  • NNMT gene Tumor cells with high methylation levels of DNA CpG sites in the region between 1050 bp before the transcription start site and 193 bp before the transcription start site have significantly stronger inhibitory effects on NNMT gene promoter region
  • NNMT gene transcription start Tumor cells with low methylation levels of DNA CpG sites in the region between the first 1050bp of the site and 499bp after the transcription start site
  • the inhibitory effect was significantly weaker, indicating that the tumor cell NNMT gene promoter region, the region between 1050bp before the NNMT gene transcription start site and 499bp after the transcription start site, and the NNMT gene transcription start site 1050bp before the transcription start site.
  • the methylation level of DNA CpG sites in the region between the first 193 bp and the susceptibility to mitochondrial oxidative phosphorylation pathway inhibitors was positively correlated.
  • tumor cell lines (NCI-H82, G-401, SW48 and WSU-DLCL2) sensitive to mitochondrial oxidative phosphorylation pathway inhibitors and three tumor cell lines insensitive (786-O, CFPAC-1 and SF126)
  • bisulfite treatment is performed on the cellular genomic DNA to deaminate the unmethylated cytosine into uracil, while the methylated cytosine is not deaminated, so the heavy submerged cytosine can be deaminated based on this.
  • Sulfate-treated and untreated sequencing samples were compared to discover methylated sites, followed by PCR amplification of the region with the corresponding primers, and sequencing analysis to detect methylation levels at CpG sites within the DNA region.
  • the sites of human chromosome 11 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 correspond to the sites of the nucleotide sequence of SEQ ID NO: 1 as follows:
  • SAM S-adenosylmethionine
  • methylation donor SAM levels in the cell lines sensitive to mitochondrial oxidative phosphorylation pathway inhibitors were significantly higher than those for mitochondrial oxidative phosphorylation Levels of methylation donor SAM in cell lines insensitive to cytotoxic pathway inhibitors (786-O, CFPAC-1 and SF126).
  • the methylation level of cellular DNA is maintained by the DNA methylases DNMT3a, DNMT3b, and DNMT1.
  • DNMT3a, DNMT3b can de novo methylate DNA
  • DNMT1 can be in the protein UHRF1 (ubiquitin-like protein containing PHD and RING finger domain).
  • UHRF1 ubiquitin-like protein containing PHD and RING finger domain
  • the DNMT1 DNA Methyltransferase1 gene transcription level was investigated at the cellular level as a biomarker of sensitivity to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the DNMT1 gene transcription level and the sensitivity of these cells to Gboxin are exponentially positively correlated, indicating that the DNMT1 gene transcription level and related cells are sensitive to mitochondrial oxidative phosphorylation pathway inhibitors.
  • the sensitivity of tumor cells was positively correlated, that is, the higher the transcription level of DNMT1 gene in tumor cells, the higher the sensitivity to mitochondrial oxidative phosphorylation pathway inhibitors.
  • NNMT neuropeptide kinase
  • DNMT1 oxidative phosphorylation pathway inhibitors
  • the NNMT gene was introduced into NCI-H82 cells through a viral vector, so that NCI-H82 cells overexpressed NNMT protein, and the expression of DNMT1 in NCI-H82 cells was knocked down by transfection of shRNA, and the intracellular ATP level was detected.
  • the experimental results are shown in Figure 21 and Figure 22. Control group of NCI-H82 cells infected with empty virus.
  • DNMT1 expression of cells (sh-DNMT1#1 in the figure is a kind of shRNA against DNMT1 gene, its corresponding DNA sequence is: GATCCGGCCCAATGAGACTGACATCAATTCAAGAGATTGATGTCAGTCTCATTGGGCTTTTTG (SEQ ID No: 2), sh-DNMT1#2 is another kind of shRNA against DNMT1 gene.
  • the NNMT protein content of NCI-H82 (ov-NNMT) overexpressing NNMT protein compared with normal NCI-H82 (Vector) detected by Western Blot experiment is shown in Figure 23.
  • Western Blot assay detected the DNMT1 protein content of NCI-H82 (sh-DNMT1#1 or sh-DNMT1#2-DNMT1) in two shRNA knockdown tumor cells compared with normal NCI-H82 (shVector) As shown in Figure 24.
  • S-Gboxin was used in sensitive cells (NCI-H82) and insensitive cells (CFPAC-1) Efficacy testing was performed on subcutaneous tumors of inoculated mice.
  • the compound S-Gboxin can significantly inhibit the subcutaneous tumor of nude mice inoculated with sensitive cells NCI-H82, and has a significant inhibitory effect on the subcutaneous tumor of nude mice inoculated with NCI-H82-NNMT ov
  • the inhibitory effect on subcutaneous tumors of nude mice inoculated with NCI-H82 was worse than that in nude mice inoculated with NCI-H82, and S-Gboxin had no obvious inhibitory effect on subcutaneous tumors in nude mice inoculated with insensitive cells CFPAC-1, indicating that oxidative phosphorylation pathway inhibitors had low NNMT expression on tumors.
  • the inhibitory effect of NNMT is stronger, that is, tumors with low NNMT expression are more sensitive to oxidative phosphorylation pathway inhibitors, and tumors with high NNMT expression are less sensitive to oxidative phosphorylation pathway inhibitors.

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Abstract

本发明涉及用于判断线粒体氧化磷酸化通路抑制剂抗癌效果的标志物。具体地，本发明涉及一种线粒体氧化磷酸化通路抑制剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤。本发明发现线粒体氧化磷酸化通路抑制剂对线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤具有显著优异的治疗效果。

Description

用于判断线粒体氧化磷酸化通路抑制剂抗癌效果的标志物 技术领域
本发明涉及药物领域，具体地涉及用于判断线粒体氧化磷酸化通路抑制剂抗癌效果的标志物。
背景技术
线粒体在真核细胞中普遍存在，为细胞的生命活动提供能量和其他细胞生长必需的中间产物。线粒体作为细胞内的能量工厂和原料来源，也是肿瘤细胞成瘤不可或缺的细胞器，抑制线粒体功能能有效抑制肿瘤的发生发展，降低病人肿瘤的恶性程度，增加病人的生存期。
氧化磷酸化通路(Oxidative Phospholytion,OXPHOS)是线粒体最重要的通路之一，该通路利用三羧酸循环和脂肪氧化等通路来源的NADH和FADH等来合成ATP。线粒体氧化磷酸化通路有90余个蛋白组成，这些蛋白分别组成5个蛋白复合体，复合体I、II、III、IV和V。前4个蛋白复合体(复合体I、II、III和IV)又称为电子传递链，它们从电子供体NADH和FADH得到电子并将其传递给氧气。在传递电子的过程中，氢离子从线粒体内膜内侧泵到线粒体内膜和线粒体外膜间的膜间腔，从而在内膜内外形成氢离子梯度和电势差。储存于线粒体膜电势中的能量驱动氧化磷酸化通路中的复合体V，从而产生ATP。恶性程度较高的具有干细胞特性的癌细胞极为依赖该通路存活，抑制该通路可有效杀伤此类癌细胞，从而可解决相关恶性癌症复发问题，此类肿瘤能量代谢类药物是新一代抗癌药物研发的重要方向。
近年来发现一些可有效靶向线粒体氧化磷酸化通路的小分子，这些小分子在多种肿瘤模型和临床实验中取得明显抗癌效果，特别是对一些复发或转移的恶性癌症效果更好，能够为未满足的临床需求提供有效的解决方案。如发表在《Nature》的一项研究发现胰腺癌中引起其复发的肿瘤细胞对氧化磷酸化通路抑制剂Oligomycin非常敏感，此类抑制剂可有效杀伤肿瘤细胞，阻断肿瘤复发，参见Viale,A.,Pettazzoni,P.,Lyssiotis,C.et al.Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.Nature 514,628–632(2014)；同样发表在《Nature》的另一项研究发现线粒体氧化磷酸化通路抑制剂Gboxin对脑瘤等多种肿瘤有较好杀伤作用，可有效抑制肿瘤生长，参见Shi,Y.,Lim,S.K.,Liang,Q.et al.Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma.Nature 567,341–346(2019)；发表在《Nature Medicine》的一项研究发现线粒体氧化磷酸化通路抑制剂IACS-010759对脑瘤和急性骨髓性白血病等有很好抑制效果，参见Molina,J.R.,Sun,Y.,Protopopova,M.et al.An inhibitor of oxidative phosphorylation exploits cancer vulnerability.Nat Med 24,1036–1046(2018)。
同时有各种研究及临床案例发现，各类型抗肿瘤药物并不会对所有肿瘤都有效，某些肿瘤细胞对特定抗肿瘤药物不敏感，特定抗肿瘤药物用在对其不敏感的肿瘤细胞或肿瘤病人上不但起不到较好治疗效果，反而会贻误治疗时机，对肿瘤病人有很大的负面影响。特别是截至目前，针对线粒体氧化磷酸化通路抑制剂这类新颖抗癌药物的特定肿瘤标记物还知之甚少。
然而，当前对相关肿瘤细胞对氧化磷酸化通路抑制剂敏感的机制还不清楚，与氧化 磷酸化通路相关的肿瘤标记物还未见报道。相关肿瘤标记物的发现能够为线粒体氧化磷酸化通路抑制剂这类特定抗肿瘤药物提供精确的用药指引，从而实现对癌症患者的精准化治疗，可显著提癌症患者的临床治疗效果，并可避免对不适合使用此类特定抗癌药物的患者用药，贻误其治疗时机。因此，本领域亟需开发一种能够有效指引线粒体氧化磷酸化抑制剂这类抗肿瘤药物使用的生物标志物，从而精确指导该类药物用药，显著提升其治疗效果。
发明内容
本发明所述的目的在于提供一种线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物，从而对肿瘤进行精准治疗。线粒体氧化磷酸化通路抑制剂对线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤具有显著优异的治疗效果。
本发明第一方面，提供一种线粒体氧化磷酸化通路抑制剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤。
在另一优选例中，所述的肿瘤为人源肿瘤。
在另一优选例中，所述的肿瘤为人肿瘤。
在另一优选例中，所述的肿瘤包括线粒体氧化磷酸化通路上调的肿瘤。
在另一优选例中，所述线粒体氧化磷酸化通路上调是指某一细胞(如肿瘤细胞)的线粒体氧化磷酸化通路表达水平或活性大于同一细胞或正常细胞(如癌旁组织细胞)中线粒体氧化磷酸化通路表达水平或活性。
在另一优选例中，所述线粒体氧化磷酸化通路上调包括线粒体氧化磷酸化通路表达水平或活性高。
在另一优选例中，所述线粒体氧化磷酸化通路上调是指某一细胞(如肿瘤细胞)的线粒体氧化磷酸化通路表达水平或活性H1与同一细胞或正常细胞(如癌旁组织细胞)中的线粒体氧化磷酸化通路表达水平或活性H0的比值(H1/H0)＞1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述的同一细胞是指线粒体氧化磷酸化通路正常表达或活性正常的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但线粒体氧化磷酸化通路正常表达或活性正常的细胞。
在另一优选例中，所述的正常细胞是指线粒体氧化磷酸化通路正常表达或活性正常的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，所述的肿瘤包括NNMT基因低表达或未表达的肿瘤。
在另一优选例中，所述的NNMT基因为人源NNMT基因。
在另一优选例中，所述的NNMT基因为人NNMT基因。
在另一优选例中，所述的肿瘤包括DNA甲基化酶高表达的肿瘤。
在另一优选例中，所述的DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b， 或其组合。
在另一优选例中，所述的肿瘤包括DNMT1高表达的肿瘤。
在另一优选例中，所述的肿瘤包括DNMT3a高表达的肿瘤。
在另一优选例中，所述的肿瘤包括DNMT3b高表达的肿瘤。
在另一优选例中，所述的肿瘤包括UHRF1高表达的肿瘤。
在另一优选例中，所述肿瘤包括NNMT基因核苷酸位点甲基化水平高和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
在另一优选例中，所述肿瘤包括NNMT基因核苷酸位点甲基化水平高的肿瘤。
在另一优选例中，所述肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
在另一优选例中，所述NNMT基因低表达或未表达的肿瘤是指从该肿瘤中提取的1μg蛋白中通过NNMT抗体检测不到NNMT蛋白，更佳地是5μg，更佳地是10μg，更佳地是100μg，更佳地是1000μg。
在另一优选例中，所述NNMT基因低表达或未表达的肿瘤是指肿瘤细胞的NNMT基因的表达水平小于同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达水平。
在另一优选例中，所述NNMT基因低表达或未表达的肿瘤是指肿瘤细胞的NNMT基因的表达水平E1与同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达水平E0的比值(E1/E0)<1.0。
在另一优选例中，所述NNMT基因低表达或未表达是指某一细胞(如肿瘤细胞)的NNMT基因的表达E1与同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达E0的比值(E1/E0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
在另一优选例中，所述的同一细胞是指NNMT基因正常表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但NNMT基因正常表达的细胞。
在另一优选例中，所述的正常细胞是指NNMT基因正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，E0为NNMT基因正常表达细胞的NNMT基因的表达水平。
在另一优选例中，所述的NNMT基因正常表达的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述DNA甲基化酶高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶，更佳地是5μg，更佳地是1μg，更佳地是0.2μg，更佳地是0.05μg，更佳地是0.01μg。
在另一优选例中，所述DNA甲基化酶高表达的肿瘤是指肿瘤细胞的DNA甲基化酶的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNA甲基化酶的表达水平。
在另一优选例中，所述DNA甲基化酶高表达的肿瘤是指肿瘤细胞的DNA甲基化酶的表达水平A1与同一细胞或正常细胞(如癌旁组织细胞)中DNA甲基化酶的表达水平A0的比值(A1/A0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述的同一细胞是指DNA甲基化酶正常表达的细胞(如同一类肿瘤 细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNA甲基化酶正常表达的细胞。
在另一优选例中，所述的正常细胞是指DNA甲基化酶正常表达的正常组织细胞(如如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，A0为DNA甲基化酶正常表达细胞的DNA甲基化酶的表达水平。
在另一优选例中，所述的DNA甲基化酶正常表达的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述DNMT1高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白，更佳地是5μg，更佳地是1μg，更佳地是0.2μg，更佳地是0.05μg，更佳地是0.01μg。
在另一优选例中，所述DNMT1高表达的肿瘤是指肿瘤细胞的DNMT1的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNMT1的表达水平。
在另一优选例中，所述DNMT1高表达的肿瘤是指肿瘤细胞的DNMT1的表达水平B1与同一细胞或正常细胞(如癌旁组织细胞)中DNMT1的表达水平B0的比值(B1/B0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述的同一细胞是指DNMT1正常表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNMT1正常表达的细胞。
在另一优选例中，所述的正常细胞是指DNMT1正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，B0为DNMT1正常表达细胞的DNMT1的表达水平。
在另一优选例中，所述的DNMT1正常表达的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述DNMT3a高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白，更佳地是5μg，更佳地是1μg，更佳地是0.2μg，更佳地是0.05μg，更佳地是0.01μg。
在另一优选例中，所述DNMT3a高表达的肿瘤是指肿瘤细胞的DNMT3a的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNMT3a的表达水平。
在另一优选例中，所述DNMT3a高表达的肿瘤是指肿瘤细胞的DNMT3a的表达水C1与同一细胞或正常细胞(如癌旁组织细胞)中DNMT3a的表达水平C0的比值(C1/C0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述的同一细胞是指DNMT3a正常表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNMT3a正常表达的细胞。
在另一优选例中，所述的正常细胞是指DNMT3a正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，C0为DNMT3a正常表达细胞的DNMT3a的表达水平。
在另一优选例中，所述的DNMT3a正常表达的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述DNMT3b高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白，更佳地是5μg，更佳地是1μg，更佳地是0.2μg，更佳地是0.05μg，更佳地是0.01μg。
在另一优选例中，所述DNMT3b高表达的肿瘤是指肿瘤细胞的DNMT3b的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNMT3b的表达水平。
在另一优选例中，所述DNMT3b高表达的肿瘤是指肿瘤细胞的DNMT3b的表达水D1与同一细胞或正常细胞(如癌旁组织细胞)中DNMT3b的表达水平D0的比值(D1/D0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述的同一细胞是指DNMT3b正常表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNMT3b正常表达的细胞。
在另一优选例中，所述的正常细胞是指DNMT3b正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，D0为DNMT3b正常表达细胞的DNMT3b的表达水平。
在另一优选例中，所述的DNMT3b正常表达的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述UHRF1高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白，更佳地是5μg，更佳地是1μg，更佳地是0.2μg，更佳地是0.05μg，更佳地是0.01μg。
在另一优选例中，所述UHRF1高表达的肿瘤是指肿瘤细胞的UHRF1的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中UHRF1的表达水平。
在另一优选例中，所述UHRF1高表达的肿瘤是指肿瘤细胞的UHRF1的表达水平F1与同一细胞或正常细胞(如癌旁组织细胞)中UHRF1的表达水平F0的比值(F1/F0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述的同一细胞是指UHRF1正常表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但UHRF1正常表达的细胞。
在另一优选例中，所述的正常细胞是指UHRF1正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，F0为UHRF1正常表达细胞的UHRF1的表达水平。
在另一优选例中，所述的UHRF1正常表达的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平大于同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因核苷酸位点甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平L1与同一细胞或正常细胞(如肿瘤旁组织细胞)中NNMT基因核苷酸位点甲基化水平L0的比值(L1/L0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳 地≥30，更佳地≥50。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平≥1％，较佳地≥3％，较佳地≥5％，较佳地≥10％，较佳地≥15％,较佳地≥20％,更佳地≥25％，更佳地≥30％，更佳地≥40％，更佳地≥50％。
在另一优选例中，所述的同一细胞是指NNMT基因核苷酸位点甲基化水平为正常水平的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但NNMT基因核苷酸位点甲基化水平为正常水平的细胞。
在另一优选例中，所述的正常细胞是指NNMT基因核苷酸位点甲基化水平为正常水平的正常组织细胞(如如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，所述的NNMT基因核苷酸位点甲基化水平正常水平的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平(M％)≥3％且小于等于M1％，其中，M1为3-100之间的任一正整数。
在另一优选例中，M1为5、10、15、20、25、30、35、40、45、50、55、60、65、70、80、85、90、95或100。
在另一优选例中，所述的NNMT基因核苷酸位点甲基化水平是指NNMT基因区的甲基化的核苷酸数量占NNMT基因区所有核苷酸数量的比值。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因启动子区的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因启动子区的核苷酸序列如SEQ ID NO:1所示。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因转录起始位点前1050bp到转录起始位点后499bp区域内的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp区域内的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因转录起始位点前840bp到转录起始位点前469bp区域内的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前840bp到转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位和114166066位中任何两个位点之间的区域内(包括这两个位点本身)的核苷酸位点甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050 位和114166066位中的一个或多个(如2、3、4、5、6或7)位点的核苷酸甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括选自下组的位点的核苷酸甲基化水平：人11号染色体114165695位、人11号染色体114165730位、人11号染色体114165769位、人11号染色体114165804位、人11号染色体114165938位、人11号染色体114166050位、人11号染色体114166066位，或其组合。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括SEQ ID NO:1核苷酸序列的位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中任何两个位点之间的区域内(包括这两个位点本身)的核苷酸位点甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括SEQ ID NO:1核苷酸序列位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中的一个或多个(如2、3、4、5、6或7)位点的核苷酸甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括选自下组SEQ ID NO:1序列位点的核苷酸甲基化水平：第1161位、第1196位、第1235位、第1270位、第1404位、第1516位、第1532位，或其组合。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平大于同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因区DNA CpG位点甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平W1与同一细胞或正常细胞(如肿瘤旁组织细胞)中NNMT基因区DNA CpG位点甲基化水平W0的比值(W1/W0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平≥1％，较佳地≥3％，较佳地≥5％，较佳地≥10％，较佳地≥15％,较佳地≥20％,更佳地≥25％，更佳地≥30％，更佳地≥40％，更佳地≥50％。
在另一优选例中，所述的同一细胞是指NNMT基因区DNA CpG位点甲基化水平为正常水平的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但NNMT基因区DNA CpG位点甲基化水平为正常水平的细胞。
在另一优选例中，所述的正常细胞是指NNMT基因区DNA CpG位点甲基化水平为正常水平的正常组织细胞(如如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平正常水平的细胞包括对线粒体氧化磷酸化通路抑制剂不敏感的细胞。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平(M％)≥3％且小于等于M2％，其中，M2为3-100之间的任一正整数。
在另一优选例中，M2为5、10、15、20、25、30、35、40、45、50、55、60、65、70、80、85、90、95或100。
在另一优选例中，所述的CpG位点甲基化水平是指某基因区域甲基化的CpG核苷酸数量占该基因区域所有核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区的甲基化的CpG核苷酸数量占NNMT基因区所有核苷酸数量的比值。
在另一优选例中，所述的CpG位点甲基化水平是指某基因区域甲基化的CpG核苷酸数量占该基因区域所有CpG核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区的甲基化的CpG核苷酸数量占NNMT基因区所有CpG核苷酸数量的比值。
在另一优选例中，所述的DNA CpG位点甲基化水平是指某区域DNA已甲基化的CpG位点数量占该区域DNA的全部CpG位点数量的比值。
在另一优选例中，所述的DNA CpG位点甲基化水平是指某区域DNA已甲基化的CpG核苷酸数量占该区域DNA的所有核苷酸数量的比值。
在另一优选例中，所述的DNA CpG位点甲基化水平是指某区域DNA已甲基化的CpG核苷酸数量占该区域DNA的全部CpG核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区DNA已甲基化的CpG位点数量占NNMT基因区DNA的全部CpG位点数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区DNA已甲基化的CpG核苷酸数量占NNMT基因区DNA的全部CpG核苷酸数量的比值。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因启动子区DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因启动子区的核苷酸序列如SEQ ID NO:1所示。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因转录起始位点前1050bp到转录起始位点后499bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因转录起始位点前840bp到转录起始位点前469bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前840bp到转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位和114166066位中任何两个位点之间的区域内(包括这两个位点本身)的DNA CpG位点甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050 位和114166066位中的一个或多个(如2、3、4、5、6或7)位点的甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括选自下组的位点的甲基化水平：人11号染色体114165695位、人11号染色体114165730位、人11号染色体114165769位、人11号染色体114165804位、人11号染色体114165938位、人11号染色体114166050位、人11号染色体114166066位，或其组合。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括SEQ ID NO:1核苷酸序列位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中任何两个位点之间的区域内(包括这两个位点本身)的DNA CpG位点甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括SEQ ID NO:1核苷酸序列位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中的一个或多个(如2、3、4、5、6或7)位点的甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括选自下组的SEQ ID NO:1序列位点的甲基化水平：第1161位、第1196位、第1235位、第1270位、第1404位、第1516位、第1532位，或其组合。
在另一优选例中，所述的肿瘤选自下组：肺癌、肾癌、乳腺癌、结肠癌、直肠癌、结直肠癌、淋巴癌、白血病、胰腺癌、脑瘤、肝癌、前列腺癌、黑色素癌，或其组合。
在另一优选例中，所述的肺癌选自下组：非小细胞肺癌、小细胞肺癌、转移性肺癌，或其组合。
在另一优选例中，所述的结肠癌包括结肠腺癌。
在另一优选例中，所述的直肠癌包括直肠腺癌。
在另一优选例中，所述的结直肠癌包括结直肠腺癌。
在另一优选例中，所述的淋巴癌选自下组：B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴癌、大细胞淋巴癌、组织细胞性淋巴癌，或其组合。
在另一优选例中，所述的淋巴癌包括弥漫大B细胞淋巴瘤。
在另一优选例中，所述的脑瘤选自下组：胶质母细胞瘤、神经胶质细胞瘤，或其组合。
在另一优选例中，所述的胶质母细胞瘤包括多形性胶质母细胞瘤。
在另一优选例中，所述的脑瘤包括髓母细胞瘤。
在另一优选例中，所述的肾癌选自下组：肾透明细胞腺癌、转移性肾癌，或其组合。
在另一优选例中，所述的肾癌的癌细胞包括肾癌Wilms细胞。
在另一优选例中，所述的白血病选自下组：T淋巴细胞白血病、髓细胞性白血病，或其组合。
在另一优选例中，所述的T淋巴细胞白血病包括急性T淋巴细胞白血病。
在另一优选例中，所述的髓细胞性白血病包括急性髓细胞性白血病。
在另一优选例中，所述的髓细胞性白血病包括AML急性髓细胞性白血病。
在另一优选例中，所述的髓细胞性白血病包括M4级AML急性髓细胞性白血病。
在另一优选例中，所述的髓细胞性白血病包括FAB M4级AML急性髓细胞性白血病
在另一优选例中，所述的表达包括蛋白表达和/或mRNA表达。
在另一优选例中，所述的前列腺癌选自下组：转移性前列腺癌。
在另一优选例中，所述的转移性前列腺癌选自下组：脑转移前列腺癌、骨转移前列腺 癌、或其组合。
在另一优选例中，所述的乳腺癌选自下组：乳腺导管癌、转移性乳腺癌，或其组合。
在另一优选例中，所述的乳腺导管癌包括原发性乳腺导管癌。
在另一优选例中，所述的乳腺导管癌包括3级原发性乳腺导管癌。
在另一优选例中，所述的胰腺癌包括肝转移胰腺癌。
在另一优选例中，所述的线粒体氧化磷酸化通路抑制剂包括式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐；
Figure PCTCN2021121088-appb-000001
其中，
R 1、R 2、R 3、R 4、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
R 5为无、氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
Z 1为
Figure PCTCN2021121088-appb-000002
其中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基；
所述的杂环烷基、杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
在另一优选例中，R5为无，
Figure PCTCN2021121088-appb-000003
为双键。
在另一优选例中，R5不为无，
Figure PCTCN2021121088-appb-000004
为单键。
在另一优选例中，R5不为无且
Figure PCTCN2021121088-appb-000005
为双键，并且与R5相连的N原子为N +。
在另一优选例中，R5为无、氢或C1-C3烷基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和R 9各自独立地为氢、卤素、 羟基、巯基、氨基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C1-C10烷氧基、取代或未取代的C1-C10烷硫基、取代或未取代的C6-C10芳基、取代或未取代的5-10元杂芳基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C8烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C1-C8烷氧基、取代或未取代的C1-C8烷硫基、取代或未取代的C6-C8芳基、取代或未取代的5-8元杂芳基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C6烷基、取代或未取代的C5-C8环烷基、取代或未取代的5-8元杂环烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C6-C8芳基、取代或未取代的5-8元杂芳基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C1-C4烷硫基、取代或未取代的C6-C8芳基、取代或未取代的5-8元杂芳基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C4烷基、取代或未取代的C5-C8环烷基、取代或未取代的5-8元杂环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C1-C4烷硫基、取代或未取代的C6-C8芳基、取代或未取代的5-8元杂芳基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C4烷基、取代或未取代的C5-C8环烷基、取代或未取代的5-8元杂环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C1-C4烷硫基、取代或未取代的C6芳基、取代或未取代的C7芳基、取代或未取代的C8芳基、取代或未取代的5-8元(例如5、6、7、8)杂芳基。
在另一优选例中，R 1、R 2、R 3、R 4、R 7和R 8各自独立地为氢。
在另一优选例中，R 5为氢、甲基、乙基、丙基、或丁基。
在另一优选例中，R 6为氢、甲基、乙基、丙基、丁基、苯基、三氟甲基-苯基-。
在另一优选例中，三氟甲基-苯基为为单取代的三氟甲基-苯基-。
在另一优选例中，三氟甲基-苯基中，三氟甲基为苯环的邻位、间位或对位取代。
在另一优选例中，三氟甲基-苯基为：
Figure PCTCN2021121088-appb-000006
在另一优选例中，R 6为氢、甲基、乙基、丙基、丁基、未取代的苯基、取代的苯基。
在另一优选例中，取代的苯基是指苯基的一个或多个(如2、3或4个)氢被三氟甲基取代。
在另一优选例中，取代的苯基是指苯基的一个或多个(如2、3或4个)氢被三氟甲基取代。
在另一优选例中，取代的苯基是指苯基的一个氢被三氟甲基取代。
在另一优选例中，取代的苯基是指苯基的一个邻位、间位或对位的氢被三氟甲基取代。
在另一优选例中，R 6为氢、甲基、乙基、丙基、丁基、或
Figure PCTCN2021121088-appb-000007
其中，R 10、R 11、R 12、R 13和R 14各自独立地为氢、C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基。
在另一优选例中，R 10、R 11、R 12、R 13和R 14各自独立地为氢、C1-C6烷基、C3-C8环烷基、C1-C6卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C6烷氧基、C1-C6烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C6卤代烷氧基、C1-C6卤代烷硫基、C6-C10芳基、5-8元杂芳基。
在另一优选例中，R 10、R 11、R 12、R 13和R 14各自独立地为氢、C1-C4烷基、C3-C8环烷基、C1-C4卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C4烷氧基、C1-C6烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C4卤代烷氧基、C1-C4卤代烷硫基、C6-C10芳基、5-8元杂芳基。
在另一优选例中，R 10、R 11、R 12、R 13和R 14各自独立地为氢、C1-C4卤代烷基(如三氟甲基)。
在另一优选例中，R 10、R 11、R 12、R 13和R 14各自独立地为氢、三氟甲基。
在另一优选例中，R 10、R 11、R 12和R 14各自独立地为氢。
在另一优选例中，R 13为三氟甲基。
在另一优选例中，Z 1为
Figure PCTCN2021121088-appb-000008
在另一优选例中，Z 1为
Figure PCTCN2021121088-appb-000009
在另一优选例中，R 9为取代或未取代的环已基。
在另一优选例中，所述取代的环已基是指环已基的一个或多个(如2、3或4个)氢各自独立地被C1-C4的烷基取代。
在另一优选例中，所述取代的环已基是指环已基的一个或多个(如2、3或4个)氢各自独立地被甲基、乙基、丙基、丁基取代。
在另一优选例中，所述取代的环已基是指环已基的1位和4位的氢被被C1-C4烷基取代。
在另一优选例中，所述取代的环已基是指环已基的1位和4位的氢被被甲基、乙基、丙基、丁基取代取代。
在另一优选例中，R 9为1-丙基-4-甲基-环已基-。
在另一优选例中，R 9为1-异丙基-4-甲基-环已基-。
在另一优选例中，R 9为
Figure PCTCN2021121088-appb-000010
其中，R 15、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23和R 24各自独立地为氢、C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基。
在另一优选例中，R 15、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23和R 24各自独立地为氢、C1-C6烷基、C3-C8环烷基、C1-C6卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C6烷氧基、C1-C6烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C6卤代烷氧基、C1-C6卤代烷硫基、C6-C10芳基、5-10元杂芳基。
在另一优选例中，R 15、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23和R 24各自独立地为氢、C1-C4烷基、C3-C8环烷基、C1-C4卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C4烷氧基、C1-C4烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C4卤代烷氧基、C1-C4卤代烷硫基、C6-C10芳基、5-10元杂芳基。
在另一优选例中，R 15、R 16、R 17、R 18、R 19、R 20、R 21、R 22、R 23和R 24各自独立地为氢、甲基、乙基、丙基、丁基。
在另一优选例中，丙基为异丙基。
在另一优选例中，R 9为
Figure PCTCN2021121088-appb-000011
其中，R 16、R 17、R 18、R 19、R 20、R 22、R 23和R 24如上所定义。
在另一优选例中，R 9为
Figure PCTCN2021121088-appb-000012
在另一优选例中，R 9为
Figure PCTCN2021121088-appb-000013
在另一优选例中，所述的杂环烷基、杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
在另一优选例中，当R 5为无，所述的式I化合物的结构如下式I-1所示：
Figure PCTCN2021121088-appb-000014
其中，R 1、R 2、R 3、R 4、R 6、R 7、R 8、R 9和Z1如上所定义。
在另一优选例中，当R 5不为无时，式I化合物的一种盐的结构如下式I-2所示：
Figure PCTCN2021121088-appb-000015
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和Z1如上所定义。
在另一优选例中，所述的式I化合物具有如下式I-3结构：
Figure PCTCN2021121088-appb-000016
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和
Figure PCTCN2021121088-appb-000017
如上所定义。
在另一优选例中，所述的线粒体氧化磷酸化通路抑制剂包括式II化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐；
Figure PCTCN2021121088-appb-000018
其中，
R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35和R 36各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C1-C12卤代烷氧基、取代或未取代的C1-C12卤代烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
Z 2和Z 3各自独立地为取代或未取代的C6-C12亚芳基、取代或未取代的3-12元亚杂芳基；
n为0、1、2、3、4、5、6、7、8、9、10、11或12；
所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基；
所述的杂环烷基、杂芳基、亚芳基、亚杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
在另一优选例中，R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35和R 36各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C1-C10烷氧基、取代或未取代的C1-C10烷硫基、取代或未取代的C1-C10卤代烷氧基、取代或未取代的C1-C10卤代烷硫基、取代或未取代的C6-C10芳基、取代或未取代的5-10元杂芳基。
在另一优选例中，R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35和R 36各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C8烷基、取代或未取代的C3-C10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C1-C8烷氧基、取代或未取代的C1-C8烷硫基、取代或未取代的C1-C8卤代烷氧基、取代或未取代的C1-C8卤代烷硫基、取代或未取代的C6-C10芳基、取代或未取代的5-10元杂芳基。
在另一优选例中，R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35和R 36各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C6烷基、取代或未取代的C3-C10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C1-C6卤代烷氧基、取代或未取代的C1-C6卤代烷硫基、取代或未取代的C6-C10芳基、取代或未取代的5-10元杂芳基。
在另一优选例中，R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35和R 36各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C1-C4烷硫基、取代或未取代的C1-C4卤代烷氧基、取代或未取代的C1-C4卤代烷硫基、取代或未取代的C6-C8芳基、取代或未取代的5-8元杂芳基。
在另一优选例中，R 25、R 26、R 28、R 29、R 30、R 31、R 32、R 34、R 35和R 36各自独立地为氢。
在另一优选例中，R 27为取代或未取代的C1-C4卤代烷氧基、取代或未取代的C1-C4卤代烷硫基。
在另一优选例中，R 27为取代或未取代的C1-C3卤代烷氧基、取代或未取代的C1-C3卤代烷硫基。
在另一优选例中，R 27为取代或未取代的C1-C2卤代烷氧基、取代或未取代的C1-C2卤代烷硫基。
在另一优选例中，R 27为三氟甲基-O-、三氟甲基-S-、
在另一优选例中，R 33为取代或未取代的3-10元(例如5、6、7、8、9、10)元杂环烷基。
在另一优选例中，所述的杂环烷基为全饱和的杂环烷基。
在另一优选例中，R 33为取代或未取代的六氢吡啶基。
在另一优选例中，R 33为取代或未取代的六氢吡啶基，所述的取代是指六氢吡啶基的一个或多个(如2、3、4、5或6个)氢各自独立地被选自下组的取代基所取代：甲磺酰基、磺酰基。
在另一优选例中，R 33为
Figure PCTCN2021121088-appb-000019
其中，
R 37、R 38、R 39、R 40、R 41、R 42、R 43、R 44、R 45和R 46各自独立地为氢、C1-C4烷基、C3-C6环烷基、甲磺酰基、磺酰基。
在另一优选例中，R 37、R 38、R 39、R 40、R 41、R 43、R 44、R 45和R 46各自独立地为氢。
在另一优选例中，R 42为甲磺酰基、磺酰基。
在另一优选例中，n为0、1、2、3、4、5、6、7或8。
在另一优选例中，n为1。
在另一优选例中，Z 2和Z 3各自独立地为取代或未取代的C6-C10亚芳基、取代或未取代的3-10元亚杂芳基。
在另一优选例中，Z 2和Z 3各自独立地为取代或未取代的C6-C8亚芳基、取代或未取代的3-8元亚杂芳基。
在另一优选例中，Z 2和Z 3各自独立地为取代或未取代的C6-C8亚芳基、取代或未取代的3-7元亚杂芳基。
在另一优选例中，Z 2和Z 3各自独立地为取代或未取代的C6亚芳基、取代或未取代的C7亚芳基、取代或未取代的C8亚芳基、取代或未取代的3元亚杂芳基、取代或未取代的4元亚杂芳基、取代或未取代的5元亚杂芳基、取代或未取代的6元亚杂芳基、取代或未取代的7元亚杂芳基、取代或未取代的8元亚杂芳基、取代或未取代的9元亚杂芳基、取代或未取代的10元亚杂芳基。
在另一优选例中，Z 2和Z 3各自独立地为亚苯基、取代或未取代的亚恶二唑基、取代或未取代的亚三氮唑基。
在另一优选例中，Z 2为取代或未取代的亚恶二唑基。
在另一优选例中，亚恶二唑基为亚1,2,4-恶二唑基、
在另一优选例中，亚三氮唑基为亚1H-1,2,4-三氮唑基。
在另一优选例中，Z 2和Z 3各自独立地为
Figure PCTCN2021121088-appb-000020
其中，R 47为氢、C1-C8烷基、C3-C8环烷基。
在另一优选例中，R 47为氢、C1-C8烷基、C3-C8环烷基。
在另一优选例中，R 47为氢、C1-C6烷基、C3-C8环烷基。
在另一优选例中，R 47为氢、C1-C4烷基、C3-C8环烷基。
在另一优选例中，R 47为氢、C1-C2烷基、C3-C8环烷基。
在另一优选例中，R 47为氢、甲基、乙基、丙基、或丁基。
在另一优选例中，Z 2为
Figure PCTCN2021121088-appb-000021
在另一优选例中，Z 3为
Figure PCTCN2021121088-appb-000022
其中，R 47如上所定义。
在另一优选例中，所述的杂环烷基、杂芳基、亚芳基、亚杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
在另一优选例中，所述的式II化合物具有如下式II-1结构：
Figure PCTCN2021121088-appb-000023
Figure PCTCN2021121088-appb-000024
其中，R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35、R 36、R 47和n如上所定义。
在另一优选例中，所述的线粒体氧化磷酸化通路抑制剂包括式III化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐；
Figure PCTCN2021121088-appb-000025
其中，
R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立地为氢、卤素、羟基、羟基-(C1-C12烷基)-、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基；
所述的杂环烷基、杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
在另一优选例中，R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立地为氢、卤素、羟基、羟基-(C1-C10烷基)-、巯基、氨基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的3-10元杂环烷基、取代或未取代的C1-C10烷氧基、取代或未取代的C1-C10烷硫基、取代或未取代的C6-C10芳基、取代或未取代的5-10元杂芳基。
在另一优选例中，R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立 地为氢、卤素、羟基、羟基-(C1-C8烷基)-、巯基、氨基、取代或未取代的C1-C8烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C1-C8烷氧基、取代或未取代的C1-C8烷硫基、取代或未取代的C6-C10芳基、取代或未取代的5-10元杂芳基。
在另一优选例中，R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立地为氢、卤素、羟基、羟基-(C1-C6烷基)-、巯基、氨基、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基。
在另一优选例中，R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立地为氢、羟基、羟基-(C1-C4烷基)-、取代或未取代的C1-C4烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C1-C4烷硫基。
在另一优选例中，R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立地为氢、甲基、乙基、丙基、丁基、羟基-丙基-、巯基-丙基-、羟基、巯基。
在另一优选例中，羟基-丙基-为单羟基-丙基-。
在另一优选例中，羟基-丙基-为
Figure PCTCN2021121088-appb-000026
另一优选例中，巯基-丙基-为单巯基-丙基-。
在另一优选例中，巯基-丙基-为
Figure PCTCN2021121088-appb-000027
在另一优选例中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C6烷基、C3-C8环烷基、C1-C6卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C6烷氧基、C1-C6烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C6卤代烷氧基、C1-C6卤代烷硫基、C6-C10芳基、5-10元杂芳基、甲磺酰基、磺酰基。
在另一优选例中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C4烷基、C3-C8环烷基、C1-C4卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C4烷氧基、C1-C4烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C4卤代烷氧基、C1-C4卤代烷硫基、C6-C10芳基、5-10元杂芳基、甲磺酰基、磺酰基。
在另一优选例中，所述的杂环烷基、杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
在另一优选例中，所述的线粒体氧化磷酸化通路抑制剂选自下组：
Figure PCTCN2021121088-appb-000028
在另一优选例中，所述的线粒体氧化磷酸化通路抑制剂选自下组：
Figure PCTCN2021121088-appb-000029
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的组合物或制剂还包括药学上可接受的载体。
在另一优选例中，所述的表达为mRNA或蛋白表达。
在另一优选例中，所述的组合物或制剂的剂型为固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂
在另一优选例中，所述的组合物或制剂的剂型为片剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
本发明第二方面，提供一种用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物，所述的标志物包括线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平。
本发明还提供了所述标志物(或其表达水平、或活性或甲基化水平)或其检测试剂的用途，它被用于制备用于一试剂盒，所述试剂盒用于断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平包括NNMT基因启动子区DNA CpG位点甲基化水平。
在另一优选例中，当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，所述肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂包括肿瘤患者的肿瘤对线粒体氧化磷酸化通路抑制剂敏感。
在另一优选例中，所述肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂包括肿瘤患者的肿瘤对线粒体氧化磷酸化通路抑制剂不敏感。
在另一优选例中，所述的DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合。
在另一优选例中，所述线粒体氧化磷酸化通路上调的肿瘤如本发明第一方面所述。
在另一优选例中，所述NNMT基因低表达或未表达的肿瘤如本发明第一方面所述。
在另一优选例中，所述DNA甲基化酶(如DNMT1)高表达的肿瘤如本发明第一方面所述。
在另一优选例中，所述UHRF1高表达的肿瘤如本发明第一方面所述。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高的肿瘤如本发明第一方面所述。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高的肿瘤如本发明第一方面所述。
在另一优选例中，所述线粒体氧化磷酸化通路下调是指某一细胞(如肿瘤细胞)的线粒体氧化磷酸化通路表达水平或活性H1与同一细胞或正常细胞(如癌旁组织细胞)中的线粒体氧化磷酸化通路表达水平或活性H0的比值(H1/H0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
在另一优选例中，所述所述NNMT基因高表达是指某一细胞(如肿瘤细胞)的NNMT基因的表达E1与同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达E0的比值 (E1/E0)＞1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50。
在另一优选例中，所述DNA甲基化酶低表达的肿瘤是指肿瘤细胞的DNA甲基化酶的表达水平A1与同一细胞或正常细胞(如癌旁组织细胞)中DNA甲基化酶的表达水平A0的比值(A1/A0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
在另一优选例中，所述UHRF1低表达的肿瘤是指肿瘤细胞的UHRF1的表达水F1与同一细胞或正常细胞(如癌旁组织细胞)中UHRF1的表达水平F0的比值(F1/F0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平低是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平L1与同一细胞或正常细胞(如肿瘤旁组织细胞)中NNMT基因核苷酸位点甲基化水平L0的比值(L1/L0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平低是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平W1与同一细胞或正常细胞(如肿瘤旁组织细胞)中NNMT基因区DNA CpG位点甲基化水平W0的比值(W1/W0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
本发明第三方面，提供一种检测试剂盒，所述的检测试剂盒包括：
(i)用于检测线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂。
在另一优选例中，所述检测试剂盒的检测样本包括肿瘤细胞。
在另一优选例中，NNMT基因表达是指该基因mRNA和该蛋白的表达；
在另一优选例中，NNMT基因区DNA CpG位点甲基化水平是指NNMT基因启动子区DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因区DNA CpG位点甲基化水平是指NNMT基因转录起始位点前1050bp到基因转录起始位点后499bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因区DNA CpG位点甲基化水平是指NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因区DNA CpG位点甲基化水平是指NNMT基因转录起始位点前840bp到转录起始位点前469bp区域内DNA CpG位点甲基化水平。
本发明第四方面，提供一种如发明第三方面所述的检测试剂盒的用途，用于制备一伴随诊断试剂盒，所述伴随诊断试剂盒用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，所述的伴随诊断试剂盒还包括说明书或标签
在另一优选例中，所述的说明书或标签记载：
当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，所述的说明书或标签记载：当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
本发明第五方面，提供一种药盒，所述的药盒包括：
(i)用于检测线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂；和
(ii)线粒体氧化磷酸化通路抑制剂。
在另一优选例中，所述的药盒还包括说明书或标签。
在另一优选例中，所述的说明书或标签记载：
当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，该肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNACpG位点甲基化水平低，则该肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
本发明第六方面，提供一种预防和/或治疗肿瘤的方法，给所需的对象施用线粒体氧化磷酸化通路抑制剂。
在另一优选例中，所述对象的肿瘤包括NNMT基因低表达或未表达的肿瘤。
在另一优选例中，所述对象的肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
在另一优选例中，所述对象为人和非人哺乳动物(啮齿动物、兔、猴、家畜、狗、猫等)。
本发明第七方面，提供一种装置或系统，所述的装置或系统包括：
(i)检测模块，所述的检测模块用于检测线粒体氧化磷酸化通路表达水平或活性、 NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平；
(ii)输出模块，所述的输出模块包括输出以下信息：
当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗；和/或
当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
在另一优选例中，所述的装置包括基因检测仪或蛋白检测仪。
在另一优选例中，所述的装置或系统还包括进样模块。
在另一优选例中，所述的进样模块用于进肿瘤细胞提取物。
在另一优选例中，所述的装置或系统还包括数据处理模块。
在另一优选例中，所述的数据处理模块处理得到线粒体氧化磷酸化通路表达水平或活性高低、NNMT基因表达高低、DNA甲基化酶表达高低、UHRF1表达高低、NNMT基因核苷酸位点甲基化水平高低、和/或NNMT基因区DNA CpG位点甲基化水平高低。
在另一优选例中，所述的数据处理模块处理得到NNMT基因表达高低和/或NNMT基因启动子区DNA CpG位点甲基化水平高低。
在另一优选例中，所述的数据处理模块处理得到NNMT基因表达高低和/或NNMT基因转录起始位点前1050bp到基因转录起始位点后499bp区域内DNA CpG位点甲基化水平高低。
在另一优选例中，所述的数据处理模块处理得到NNMT基因表达高低和/或NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp区域内DNA CpG位点甲基化水平高低。
在另一优选例中，所述的数据处理模块处理得到NNMT基因表达高低和/或NNMT基因转录起始位点前840bp到基因转录起始位点前469bp区域内DNA CpG位点甲基化水平。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了不同化合物对线粒体氧化磷酸化通路的抑制作用(平行重复3次)。
图2显示了线粒体氧化磷酸化通路抑制剂Gboxin和Oligomycin A小分子作用于NCI-H82、G-401和WSU-DLCL2肿瘤细胞后，肿瘤细胞中ATF4和p-s6蛋白表达情况。
图3显示了线粒体氧化磷酸化通路抑制剂Gboxin和Oligomycin A小分子作用于SF126、CFPAC-1和786-O肿瘤细胞后，肿瘤细胞中ATF4和p-s6蛋白表达情况。
图4显示了细胞的基因表达信息显示的细胞间差异程度。
图5显示了对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞的功能差异。
图6显示了对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞的代谢通路差异。
图7显示了参与表达的氧化磷酸化通路蛋白复合体。
图8显示了对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1和SF126)的线粒体膜电差。
图9显示了不同肿瘤细胞的线粒体氧消耗速率(OCR)。
图10显示了不同细胞中筛选出的表达差异显著的基因。
图11显示了肿瘤细胞的平均NNMT基因转录水平和肿瘤细胞对线粒体氧化磷酸化通路抑制剂的相关性。
图12显示了不同肿瘤细胞中的NNMT基因的mRNA和蛋白表达，上图显示了NNMT基因的mRNA表达，下图显示了NNMT基因的蛋白表达。
图13显示了不同肿瘤细胞的NNMT基因的表达和NNMT基因启动子区甲基化分析。
图14显示了对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞NNMT基因启动子区DNA CpG位点甲基化水平。
图15显示了对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域DNA CpG位点甲基化水平。
图16显示了对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域DNA CpG位点甲基化水平。
图17显示了为对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞特定NNMT基因区即人11号染色体114165695、114165730、114165769、114165804、114165938、114166050、114166066等7个位点的DNA CpG位点甲基化情况，黑点表示相关位点已甲基化，白点表示相关位点未甲基化，SST指转录起始位点，Chr11指根据GCF_000001405.25(GRCh37.p13)人类基因组版本界定的人类11号染色体。
图18显示了对线粒体氧化磷酸化通路抑制剂敏感及不敏感肿瘤细胞中的腺苷甲硫氨酸(SAM)水平。
图19显示了肿瘤细胞中NNMT的表达和DNMT1、UHRF1、DNMT3a和DNMT3b的表达的相关性。
图20显示了肿瘤细胞DNMT1基因转录水平和肿瘤细胞对线粒体氧化磷酸化通路抑制剂的相关性。
图21显示了通过转基因的方法过表达NCI-H82细胞NNMT蛋白和/或通过转染shRNA的方法敲低NCI-H82细胞DNMT1的表达后，肿瘤细胞对Gboxin的敏感性，其中，Vector为正常表达NNMT蛋白和DNMT1的NCI-H82细胞；ov-NNMT为通过转基因过表达NNMT蛋白的NCI-H82细胞；sh-DNMT1#1为通过sh-DNMT1#1敲低DNMT1表达的NCI-H82细胞，sh-DNMT1#2为通过sh-DNMT1#2敲低DNMT1表达的NCI-H82细胞，ov-NNMT/sh-DNMT1#1是同时通过转基因过表达NNMT蛋白和通过sh-DNMT1#1敲低DNMT1表达的NCI-H82细胞；ov-NNMT/sh-DNMT1#2是同时通过转基因过表达NNMT蛋白和通过sh-DNMT1#2敲低DNMT1表达的NCI-H82细胞。
图22显示了通过转基因的方法过表达NCI-H82细胞NNMT蛋白和/或通过转染shRNA的方法敲低NCI-H82细胞DNMT1的表达后，肿瘤细胞对Oligomycin A的敏感性， 其中，Vector为正常表达NNMT蛋白和DNMT1的NCI-H82细胞；ov-NNMT为通过转基因过表达NNMT蛋白的NCI-H82细胞；sh-DNMT1#1为通过sh-DNMT1#1敲低DNMT1表达的NCI-H82细胞，sh-DNMT1#2为通过sh-DNMT1#2敲低DNMT1表达的NCI-H82细胞，ov-NNMT/sh-DNMT1#1是同时通过转基因过表达NNMT蛋白和通过sh-DNMT1#1敲低DNMT1表达的NCI-H82细胞；ov-NNMT/sh-DNMT1#2是同时通过转基因过表达NNMT蛋白和通过sh-DNMT1#2敲低DNMT1表达的NCI-H82细胞。
图23显示了Western Blot实验检测相对于正常NCI-H82(Vector)相比，过表达NNMT蛋白的NCI-H82(ov-NNMT)的NNMT蛋白含量，其中，Vector为正常表达NNMT蛋白和DNMT1的NCI-H82细胞；ov-NNMT为通过转基因过表达NNMT蛋白的NCI-H82细胞。
图24显示了Western Blot实验检测相对于正常NCI-H82(shVector)相比，两种shRNA敲低肿瘤细胞的DNMT1表达的NCI-H82(sh-DNMT1#1或sh-DNMT1#2)的DNMT1蛋白含量，其中，shVector为正常表达DNMT1的NCI-H82细胞；shDNMT1#1为通过sh-DNMT1#1敲低DNMT1表达的NCI-H82细胞，shDNMT1#2为通过sh-DNMT1#2敲低DNMT1表达的NCI-H82细胞。
图25显示了氧化磷酸化通路抑制剂S-Gboxin对NCI-H82荷瘤的抑制效果，其中，NCI-H82为正常表达NNMT蛋白细胞。
图26显示了氧化磷酸化通路抑制剂S-Gboxin对NCI-H82-NNMT ov荷瘤的抑制效果，其中，NCI-H82-NNMT ov为通过转基因过表达NNMT蛋白的NCI-H82细胞。
图27显示了氧化磷酸化通路抑制剂S-Gboxin对CFPAC-1荷瘤的抑制效果。
具体实施方式
本发明人经过长期而深入的研究，首次意外地发现线粒体氧化磷酸化通路抑制剂对线粒体氧化磷酸化通路上调、NNMT基因低表达(或未表达)、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤细胞具有显著的抑制作用。线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平和/或NNMT基因启动子区DNA CpG位点甲基化水平能够作为判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物。在此基础上，发明人完成了本发明。
术语
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。如本文所用，术语“DNA CpG位点甲基化水平高”、“DNA CpG位点甲基化高水平”与“DNA CpG位点高甲基化”可互换使用。
如本文所用，术语“DNA CpG位点甲基化低水平”、“DNA CpG位点甲基化水平低”与“DNA CpG位点低甲基化”可互换使用。
如本文所用，术语“IC50”与“IC 50”可互换使用，是指半抑制浓度(50％inhibiting concentration)，即达到50％抑制效果时抑制剂的浓度。
如本文所用，术语“CpG位点甲基化”、“CpG核苷酸甲基化”与“CpG甲基化”可互换 使用。
如本文所用，“Oligomycin A”可简写为“Oligomycin”。
如本文所用，术语“P/S”是指在相关培养基中加入Penicillin(盘尼西林)以及Streptomycin(链霉素)”。
如本文所用，术语“某一细胞”指某个细胞(如某单个癌细胞)或包含多个类似细胞的一群细胞等(如某肿瘤组织)。
如本文所用，“肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂”包括肿瘤患者的肿瘤对线粒体氧化磷酸化通路抑制剂敏感。
如本文所用，“肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂”包括肿瘤患者的肿瘤对线粒体氧化磷酸化通路抑制剂不敏感。
如本文所用，“线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平”是指线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平和NNMT基因区DNA CpG位点甲基化水平中的一种或多种。
如本文所用，“线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高”是指线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高和NNMT基因区DNA CpG位点甲基化水平高中的一种或多种。
如本文所用，“线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低”是指线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低和NNMT基因区DNA CpG位点甲基化水平低中的一种或多种。
如本文所用，术语“NNMT”的英文名为Nicotinamide N-Methyltransferase。
如本文所用，术语“bp”是指base pair，碱基对。
如本文所用，术语“SST”是指转录起始位点。
如本文所用，术语“Chr11”是指GCF_000001405.25(GRCh37.p13)人类基因组版本界定的人类11号染色体。
如本文所用，“人11号染色体”是指GCF_000001405.25(GRCh37.p13)人类基因组版本界定的人类11号染色体
如本文所用，术语“转录起始位点前”、“转录起始位点后”、“转录起始位点之前”、“转录起始位点之后”均不包括转录起始位点本身。
如本文所用，术语“人11号染色体114165695位”是指人11号染色体114165695位的核苷酸；“人11号染色体114165730位”是指人11号染色体114165730位的核苷酸；“人11号染色体114165769位”是指人11号染色体114165769位的核苷酸；“人11号染色体114165804位”是指人11号染色体114165804位的核苷酸；“人11号染色体114165938位”是指人11号染色体114165938位的核苷酸；“人11号染色体114166050位”是指人11号染色体114166050位的核苷酸；“人11号染色体114166066位”是指人11号染色体 114166066位的核苷酸。
如本文所用，术语“S-腺苷甲硫氨酸”为S-adenosyl methionine，简称SAM。
如本文所用，基因表达包括该基因蛋白表达和/或该基因mRNA表达等。
如本文所用，术语“DNMT3a”是指DNA甲基转移酶3a(DNA methyltransferase 3a)。
如本文所用，术语“DNMT3b”是指DNA甲基转移酶3b(DNA methyltransferase 3b)。
如本文所用，术语“DNMT1”是指DNA甲基转移酶1(DNA methyltransferase 1)。
如本文所用，术语“UHRF1”是指泛素样含PHD和环指域蛋白1。
应当理解，本领域的普通技术人员可以选择本发明的化合物上的取代基和取代型式以产生化学上稳定的化合物，所述化合物可以通过本领域己知的技术以及下文所阐述的方法合成。如果被超过一个(多个)取代基团取代，应当理解，这多个基团可以是在同一个碳上或在不同碳上，只要产生稳定的结构即可。
如本文所用，术语“取代”或“取代的”是基团上的氢原子被非氢原子基团取代，但需要满足其化合价要求并且由取代生成化学稳定的化合物，即不会自发进行诸如环化、消除等转变的化合物。
如本文所用，“R 1”、“R1”和“R 1”的含义相同，可相互替换，其它类似定义的含义相同。
如本文所用，
Figure PCTCN2021121088-appb-000030
表示基团的连接位点。
如本文所用，术语“烷基”指只含碳原子的直链(即，无支链)或支链饱和烃基，或直链和支链组合的基团。当烷基前具有碳原子数限定(如C1-C6烷基)指所述的烷基含有1-6个碳原子，例如，C1-C4烷基指含有1-4个碳原子的烷基，代表性实例包括但不限于甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
在本发明中，术语“卤素”指F、Cl、Br或I。
在本发明中，术语“卤代”是指被卤素取代。
如本文所用，术语“卤代烷基”是指烷基的一个或多个(优选为1、2、3或4个)氢被卤素取代，所述的烷基和卤素如上所定义，当烷基前具有碳原子数限定(如C1-C8卤代烷基)指所述的烷基含有1-8个碳原子，例如，C1-C6卤代烷基指含有1-6个碳原子的卤代烷基，代表性实例包括但不限于-CF3、-CHF 2、单氟代异丙基、双氟代丁基、或类似基团。
如本文所用，术语“环烷基”指具有饱和的或部分饱和的单元环，二环或多环(稠环、桥环或螺环)环系基团。当某个环烷基前具有碳原子数限定(如C3-C12)时，指所述的环烷基具有3-12个环碳原子。在一些优选实施例中，术语“C3-C8环烷基”指具有3-8个环碳原子的饱和或部分饱和的单环或二环烷基，包括环丙基、环丁基、环戊基、环庚基、或类似基团。
如本文所用，术语“卤代环烷基”是指环烷基的一个或多个(优选为1、2、3或4个)氢被卤素取代，所述的环烷基和卤素如上所定义，当环烷基前具有碳原子数限定(如C3-C8卤代烷基)指所述的环烷基含有3-8个环碳原子，例如，C3-C8卤代烷基指含有3-6碳原子的卤代环烷基，代表性实例包括但不限于单氟代环丙基、单氯代环丁基、单氟代环戊基、双氟代环庚基，或类似基团。
术语“烷氧基”指R-O-基团，其中R为烷基，烷基为如上本文所定义，当烷氧基前具有碳原子数限定，如C1-C8烷氧基指所述的烷氧基中的烷基具有1-8个碳原子。烷氧基的代表性示例包括但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基、叔丁氧基，或类似基团。
如本文所用，术语“烷硫基”指R-S-基团，其中R为烷基，烷基为如上本文所定义，当烷硫基前具有碳原子数限定，如C1-C8烷硫基指所述的烷硫基中的烷基具有1-8个碳原子。烷硫基的代表性示例包括但不限于：甲硫基、乙硫基、正丙硫基、异丙硫基、叔丁硫基，或类似基团。
术语“环烷氧基”指R-O-基团，其中R为环烷基，环烷基为如上本文所定义，当环烷氧基前具有碳原子数限定，如C3-C8环烷氧基基指所述的环烷氧基中的环烷基具有3-8个碳原子。环烷氧基的代表性示例包括但不限于：环丙氧基、环丁氧基，或类似基团。
术语“环烷硫基”指R-S-基团，其中R为环烷基，环烷基为如上本文所定义，当环烷硫基前具有碳原子数限定，如C3-C8环烷硫基基指所述的环烷硫基中的环烷基具有3-8个碳原子。环烷硫基的代表性示例包括但不限于：环丙硫基、环丁硫基，或类似基团。
如本文所用，术语“卤代烷氧基”是指卤代烷基-O-，所述的卤代烷基如上所定，例如，C1-C6卤代烷氧基指含有1-6个碳原子的卤代烷氧基，代表性实例包括但不限于、单氟代甲氧基、单氟代乙氧基、双氟代丁氧基、或类似基团。
如本文所用，术语“卤代烷硫基”是指卤代烷基-S-，所述的卤代烷基如上所定，例如，C1-C6卤代烷硫基指含有1-6个碳原子的卤代烷硫基，代表性实例包括但不限于、单氟代甲硫基、单氟代乙硫基、双氟代丁硫基、或类似基团。
术语“杂环烷基”是指完全饱和的或部分不饱和的的环状基团(包含但不限于如3-7元单环，7-11元双环，或8-16元三环系统)，其中至少有一个杂原子存在于至少有一个碳原子的环中。当杂环烷基前有元数限定时，指的是杂环烷基的环原子个数，例如3-16元杂环烷基指的是具有3-16个环原子的杂环烷基。每个含有杂原子的杂环上可以带有一个或多个(如1，2，3或4个)杂原子，这些杂原子各自独立地选自氮原子、氧原子或硫原子，其中氮原子或硫原子可以被氧化，氮原子也可以被季铵化。杂环烷基可以连接到环或环系分子的任何杂原子或碳原子的残基上。典型的单环杂环烷基包括但不限于氮杂环丁烷基、氧杂环丁烷基、四氢呋喃基、哌啶基、哌嗪基、4-哌啶酮基、四氢吡喃基等。多环杂环烷基包括螺环、稠环和桥环的杂环基；其中涉及到的螺环、稠环和桥环的杂环烷基任选与其他基团通过单键相连接，或者通过环上的任意两个或两个以上的原子与其它环烷环、杂环进一步并环连接。
术语“芳基”指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，是一种芳香环状烃类化合物基团，当芳基前面具有碳原子数限定，如C6-C12芳基，则指所述的芳基具有6-12个环碳原子，例如苯基和萘基。
术语“亚芳基”指芳基失去一个氢原子形成的基团，所述的芳基如上所定，当亚芳基前面具有碳原子数限定，如C6-C12亚芳基，则指所述的亚芳基具有6-12个环碳原子，亚芳基的代表性实例包括但不限于亚苯基和亚萘基等。
术语“杂芳基”指具有一个到多个(优选为1、2、3或4个)杂原子的芳族杂环系基团，其可以是单环(单环的)或者稠合在一起或共价地连接的多环(二环的、三环的或多环的)，每个含有杂原子的杂环上可以带有一个多个(如1、2、3、4个)各自独立选自下组的杂原子：氧、硫和氮。当杂芳基前有元数限定时，指的是杂芳基的环原子个数，例如5-12元杂芳基指的是具有5-12个环原子的杂芳基，代表性的例子包括但不限于：吡咯基、吡唑基、咪唑基、噁唑基、异噁唑基、噻唑基、异噻唑基、呋喃基、吡啶基、吡嗪基、嘧啶基、哒嗪基、三氮唑基及四氮唑基等。
术语“亚杂芳基”指杂芳基失去一个氢原子形成的基团，所述的杂芳基如上所定，当亚杂芳基前面具有碳原子数限定，如C6-C12亚杂芳基，则指所述的亚杂芳基具有6-12个环碳原子，亚杂芳基的代表性实例包括但不限于亚吡咯基、亚吡唑基、亚咪唑基、亚三氮唑基、亚恶二唑基和亚噁唑基等等。
如本文所用，在单独或作为其他取代基一部分时，术语"甲磺酰基"表示
Figure PCTCN2021121088-appb-000031
在本说明书中，应解释为所有取代基为未取代的，除非在本文中明确描述为“取代的”。术语“取代”是指特定的基团上的一个或多个氢原子被特定的取代基所取代。特定的取代基为在前文中相应描述的取代基，或各实施例中所出现的取代基，优选地，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基。除非特别说明，某个任意取代的基团可以在该基团的任何可取代的位点上具有一个选自特定组的取代基，所述的取代基在各个位置上可以是相同或不同的。
在本发明中，术语“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。
本发明所述的“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述的线粒体氧化磷酸化通路抑制剂时观察到的水平相比，本发明所述线粒体氧化磷酸化通路抑制剂将相关疾病(如肿瘤)及其并发症减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％，或100％。
线粒体氧化磷酸化通路
氧化磷酸化通路(Oxidative Phosphorylation,OXPHOS)是线粒体最重要的通路之一，该通路利用三羧酸循环和脂肪氧化等通路来源的NADH和FADH等来合成ATP。线粒体氧化磷酸化通路有90余个蛋白组成，这些蛋白分别组成5个蛋白复合体，复合体I、II、III、IV和V。前4个蛋白复合体(复合体I、II、III和IV)又称为电子传递链，它们从电子供体NADH和FADH得到电子并将其传递给氧气。在传递电子的过程中，氢离子从线粒体内膜内侧泵到线粒体内膜和线粒体外膜间的膜间腔，从而在内膜内外形成氢离子梯度和电势差。储存于线粒体膜电势中的能量驱动氧化磷酸化通路中的复合体V，从而产生ATP。研究表明，线粒体氧化磷酸化通路对细胞生长非常重要，与许多疾病有关，例如癌症、免疫相关疾病、神经退行性疾病，抑制线粒体氧化磷酸化通路能够用于治疗癌症、免疫相关疾病、神经退行性疾病，尤其是恶性程度较高的具有干细胞特性的癌细胞极为依赖该通路存活，抑制该通路可有效杀伤此类癌细胞，从而可解决相关恶性癌症复发问题。
NNMT基因
在本发明中，NNMT英文名为Nicotinamide N-Methyltransferase,不同数据库对NNMT基因有不同的识别号：HGNC:7861；Entrez Gene:4837；Ensembl:ENSG00000166741； OMIM:600008；UniProtKB:P40261。
根据GCF_000001405.25(GRCh37.p13)人类基因组版本,NNMT基因区位于人类11号染色体第114,128,528位bp到114,184,258位bp，总长为55,731bp的DNA序列，包括NNMT基因启动子区、NNMT基因外显子区和NNMT基因内含子区，NNMT基因转录起始位点为第114,166,535位bp。
NNMT基因启动子区为人11号染色体第114,164,535位bp到114,167,034位bp的核苷酸序列，即NNMT基因转录起始位点前2000bp(粗体部分)至转录起始位点本身及其后499bp(下划线部分)之间的序列，总长为2500bp的区域为NNMT基因启动子区，NNMT基因启动子区的核苷酸序列如下面SEQ ID NO:1所示：
SEQ ID NO:1：
Figure PCTCN2021121088-appb-000032
Figure PCTCN2021121088-appb-000033
在本发明中，NNMT基因转录起始位点前1050bp到基因转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
在本发明中，NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
在本发明中，NNMT基因转录起始位点前840bp到基因转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
在本发明中，人11号染色体114165695、114165730、114165769、114165804、114165938、114166050、114166066的位点对应于SEQ ID NO:1核苷酸序列的位点如下表1所示：
表1
人11号染色体的位点 对应于SEQ ID NO:1核苷酸序列的位点
114165695位 第1161位
114165730位 第1196位
114165769位 第1235位
114165804位 第1270位
114165938位 第1404位
114166050位 第1516位
114166066位 第1532位
DNA甲基化(DNA methylation)
DNA甲基化(DNA methylation)为DNA化学修饰的一种形式，能够在不改变DNA序列的前提下，改变遗传表现。大量研究表明，DNA甲基化能引起染色质结构、DNA构象、DNA稳定性及DNA与蛋白质相互作用方式的改变，从而调控基因表达。
DNA甲基化是最早被发现、也是研究最深入的表观遗传调控机制之一。广义上的DNA甲基化是指DNA序列上特定的碱基在DNA甲基转移酶(DNA methyltransferase，DNMT)的催化作用下，以S-腺苷甲硫氨酸(S-adenosyl methionine，SAM)作为甲基供体，通过共价键结合的方式获得一个甲基基团的化学修饰过程。这种DNA甲基化修饰可以发生在胞嘧啶的C-5位、腺嘌呤的N-6位及鸟嘌呤的N-7位等位点。一般研究中所涉及的DNA甲基化主要是指发生在CpG二核苷酸中胞嘧啶上第5位碳原子的甲基化过程，其产物称为5—甲基胞嘧啶(5-mC)，是植物、动物等真核生物DNA甲基化的主要形式。DNA甲基化作为一种相对稳定的修饰状态，在DNA甲基转移酶的作用下，可随DNA的复制过程遗传给新生的子代DNA，是一种重要的表观遗传机制。
DNA甲基化反应分为2种类型。一种是2条链均未甲基化的DNA被甲基化，称为从头甲基化(denovo methylation)；另一种是双链DNA的其中一条链已存在甲基化，另一条未甲基化的链被甲基化，这种类型称为保留甲基化(maintenance methylation)。
典型地，DNA甲基化为DNA CpG位点甲基化。CpG双核苷酸在人类基因组中的分布很不均一，而在基因组的某些区段，CpG保持或高于正常概率。CpG位点富集区(又称CpG岛)主要位于基因的启动子(promotor)和外显子区域，是富含CpG二核苷酸的一些区域，约有60％以上基因的启动子含有CpG岛。这里CpG是胞嘧啶(C)—磷酸(p)—鸟嘌呤(G)的缩写。
细胞内基因表达受多种信号传导通路、转录因子和表观遗传修饰的调控。DNA甲基化修饰是表观遗传修饰调控基因表达的重要方式，特定基因区DNA甲基化水平往往影响该基因的表达水平。相对于信号传导通路和转录因子等对基因表达的调控，表观遗传修饰中的DNA甲基化修饰对基因表达的影响更加稳定，并不易被细胞外环境所影响，DNA甲基化修饰容易用现有技术精准检测，因此是较为理想的生物标志物。
线粒体氧化磷酸化通路抑制剂及其用途
本发明提供一种线粒体氧化磷酸化通路抑制剂，用于预防和/或治疗肿瘤(癌症)。
在本发明的一个优选例中，所述的线粒体氧化磷酸化通路抑制剂包括式I、式II和/或式III化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐.
Figure PCTCN2021121088-appb-000034
具体地，本发明所述的式I、式II和/或式III化合物如本发明第一方面所述。
如本文所用，“本发明式I化合物”、或“式I化合物”可互换使用，指具有式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐。应理解，该术语 还包括上述组分的混合物。
如本文所用，“本发明式II化合物”、或“式II化合物”可互换使用，指具有式II化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐。应理解，该术语还包括上述组分的混合物。
如本文所用，“本发明式III化合物”、或“式III化合物”可互换使用，指具有式III化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐。应理解，该术语还包括上述组分的混合物。
术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐，适合形成盐的酸包括(但并不限于)：盐酸、氢溴酸、氢氟酸、氢氟酸、氢碘酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。一类优选的盐是本发明化合物与碱形成的金属盐，适合形成盐的碱包括(但并不限于)：氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸钠等无机碱、氨水、三乙胺、二乙胺等有机碱。
本发明所述的如式I所示化合物可通过常规方法转化为其药学上可接受的盐，例如，可将相应的酸的溶液加入到上述化合物的溶液中，成盐完全后除去溶剂即得本发明所述化合物的相应的盐。
代表性地，所述的线粒体氧化磷酸化通路抑制剂选自下组：
Figure PCTCN2021121088-appb-000035
本发明的研究表明，本发明化合物对线粒体氧化磷酸化通路上调、NNMT基因低表达(或未表达)、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤细胞具有更显著的抑制作用，线粒体氧化磷酸化通路上调、NNMT基因低表达(或未表达)、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤细胞对本发明线粒体氧化磷酸化通路抑制剂敏感。
癌症
本发明研究表明，本发明所述的线粒体氧化磷酸化通路抑制剂能够用于预防和/或治疗肿瘤。
在本发明中，术语“肿瘤”、“癌症”、“癌”和“瘤”可互换使用。
在本发明的一个优选例中，本发明所述的肿瘤包括线粒体氧化磷酸化通路上调的肿瘤。代表性地，本发明所述线粒体氧化磷酸化通路上调的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括NNMT基因低表达或未表达的肿瘤。代表性地，本发明所述NNMT基因低表达或未表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括DNA甲基化酶高表达的肿瘤。代表性地，本发明所述DNA甲基化酶高表达的肿瘤如上本发明第一方面所述。
本发明所述的DNA甲基化酶包括(但不限于)DNMT1、DNMT3a、DNMT3b，或其组合。优选地，本发明所述的DNA甲基化酶包括DNMT1。
在本发明的一个优选例中，本发明所述的肿瘤包括DNMT1高表达的肿瘤。代表性地，本发明所述DNMT1高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括DNMT3a高表达的肿瘤。代表性地，本发明所述DNMT3a高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括DNMT3b高表达的肿瘤。代表性地，本发明所述DNMT3b高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括UHRF1(泛素样含PHD和环指域蛋白1)高表达的肿瘤。代表性地，本发明所述UHRF1高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括NNMT基因核苷酸位点甲基化水平高的肿瘤。代表性地，本发明所述NNMT基因核苷酸位点甲基化水平高的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。代表性地，本发明所述NNMT基因区DNA CpG位点甲基化水平高的肿瘤如上本发明第一方面所述。
更具体地，本发明所述的肿瘤如上本发明第一方面所述。
在本发明中，代表性的各肿瘤细胞系对应的肿瘤种类如下表2所示：
表2
肿瘤细胞系 对应的肿瘤种类
NCI-H82 人小细胞肺癌细胞
G-401 人肾癌Wilms细胞
MDA-MB-453 乳腺癌细胞
WSU-DLCL2 人弥漫大B淋巴瘤细胞
SU-DHL-2 大细胞淋巴癌细胞
OCI-AML-3 FAB M4级AML急性髓细胞性白血病
SW48 人结肠腺癌细胞
ATN-1 T细胞白血病细胞
HCC15 非小细胞肺癌细胞
OCI-LY-19 B细胞淋巴癌细胞
22RV1 前列腺癌细胞
MIA PaCa-2 胰腺癌细胞
CCRF-CEM 急性T淋巴细胞白血病细胞
HH 皮肤T细胞淋巴癌细胞
OCI-AML-5 M4级AML急性髓细胞性白血病细胞
G-402 肾癌细胞
HCC1806 乳腺癌细胞
BT-549 乳腺癌细胞
OCI-AML-4 急性骨髓性白血病细胞
H9 淋巴瘤细胞
Jurkat,Clone E6-1 T淋巴瘤细胞
G-361 黑色素瘤细胞
U-937 组织细胞性淋巴瘤细胞
SNU-398 肝细胞癌细胞
NCI-H1048 小细胞肺癌细胞
A-375 黑色素瘤细胞
D283 Med 髓母细胞瘤细胞
GAK 黑色素瘤细胞
CHL-1 黑色素瘤细胞
NCI-H1155 非小细胞肺癌细胞
LS 180 结直肠腺癌细胞
Daoy 髓母细胞瘤细胞
DU 145 脑转移前列腺癌细胞
AM-38 多形性胶质母细胞瘤细胞
HCC70 3级原发性乳腺导管癌细胞
PANC-1 胰腺癌细胞
U-87 MG 脑瘤细胞
MJ 人皮肤T淋巴细胞瘤细胞
Gp2D 人结肠癌细胞
SU.86.86 胰腺癌细胞
NCI-H2081 小细胞肺癌细胞
NCI-H1793 非小细胞肺癌细胞
ACHN 肾癌细胞
U-251 MG 神经胶质细胞瘤细胞
MDA-MB-231 乳腺癌细胞
NCI-H196 肺癌细胞
PC-3 前列腺癌细胞
OCI-M1 急性骨髓性白血病细胞
NCI-H1651 非小细胞肺癌
C3A 肝癌细胞
SNU-449 肝癌细胞
GB-1 脑胶质母细胞瘤细胞
769-P 肾癌细胞
COLO 320HSR 结直肠腺癌细胞
CFPAC-1 胰腺癌细胞
SF126 脑瘤细胞
786-O 肾透明细胞腺癌细胞
标志物
本发明还提供一种用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物，所述的标志物包括线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平。
在一个实施方式中，线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平作为判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物，其方法包括但不限于：
当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗；和/或
当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
具体地，本发明所述线粒体氧化磷酸化通路上调的肿瘤、NNMT基因低表达或未表达的肿瘤、DNA甲基化酶(如DNMT1)高表达的肿瘤、UHRF1高表达的肿瘤、NNMT基因核苷酸位点甲基化水平高的肿瘤、
和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤如上本发明第一方面所述。
具体地，本发明所述线粒体氧化磷酸化通路下调的肿瘤、NNMT基因高表达的肿瘤、DNA甲基化酶(如DNMT1)低表达的肿瘤、UHRF1低表达的肿瘤、NNMT基因核苷酸位点甲基化水平低的肿瘤、和/或NNMT基因区DNA CpG位点甲基化水平低的肿瘤如上本发明第二方面所述。
组合物或制剂、活性成分的组合和药盒和施用方法
本发明所述的组合物优选为药物组合物，本发明所述的组合物可以包括药学上可接受的载体。
如本文所用“药学上可接受的载体”是指一种或多种相容性固体、半固体、液体或凝胶填料，它们适合于人体或动物使用，而且必须有足够的纯度和足够低的毒性。“相容性”是指药物组合物中的各组分和药物的活性成分以及它们之间相互掺和，而不明显降低药效。
应理解，在本发明中，所述的药学上可接受的载体没有特别的限制，可选用本领域常用材料，或用常规方法制得，或从市场购买得到。药学可接受的载体部分例子有纤维素及 其衍生物(如甲基纤维素、乙基纤维素、羟丙甲基纤维素、羧甲基纤维素钠等)、明胶、滑石粉、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油、等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、缓冲剂、螯合剂、增稠剂、pH调节剂、透皮促进剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、抑菌剂、无热原水等。
在本发明的一个优选例中，所述的组合物或制剂的剂型为固体制剂、液体制剂或半固体制剂。
在本发明的一个优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂
代表性地，所述的组合物或制剂的剂型为片剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
药物制剂应与给药方式相匹配。本发明药剂还可与其他协同治疗剂一起使用(包括之前、之中或之后使用)。使用药物组合物或制剂时，是将安全有效量的药物施用于所需对象(如人或非人哺乳动物)，所述安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
本发明为线粒体氧化磷酸化通路抑制剂类药物提供可指导其精准用药的生物标志物，相关生物标志物可有效识别出对此类抗肿瘤药物敏感的肿瘤患者，提升其治疗效果，避免将该类药物施用于对其不敏感的肿瘤患者，从而可实现该类药物的精准化应用。
本发明首次意外通过系统研究发现线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平能够作为判断特定肿瘤细胞是否适合应用线粒体氧化磷酸化通路抑制剂治疗的标志物。
线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤对线粒体氧化磷酸化通路抑制剂药物敏感性高，即氧化磷酸化通路抑制剂药物对线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤具有优异的治疗作用。而且，DNA CpG位点甲基化水平的检测手段成熟、稳定、可靠，适合分子标记物的开发。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例
Oligomycin A、Gboxin、S-Gboxin、IACS-010759均是已知的线粒体氧化磷酸化通路 抑制剂。
Oligomycin A化合物的结构式如下：
Figure PCTCN2021121088-appb-000036
Gboxin化合物的结构式如下：
Figure PCTCN2021121088-appb-000037
S-Gboxin化合物的结构式如下：
Figure PCTCN2021121088-appb-000038
IACS-010759化合物的结构式如下：
Figure PCTCN2021121088-appb-000039
Figure PCTCN2021121088-appb-000040
DNMT3a是指DNA甲基转移酶3a，NCBI entrez gene:1788；Uniprotkb/Swiss-port:Q9Y6K1。
DNMT3b是指DNA甲基转移酶3b，NCBI entrez gene:1789；Uniprotkb/Swiss-port:Q9UBC3。
DNMT1是指DNA甲基转移酶1，NCBI entrez gene:1786；Uniprotkb/Swiss-port:P26358。
UHRF1是指泛素样含PHD和环指域蛋白1，NCBI entrez gene:29128；Uniprotkb/Swiss-port:Q96T88。
NNMT基因英文名为Nicotinamide N-Methyltransferase。
NNMT基因启动子区核苷酸序列如SEQ ID NO:1所示。
NNMT基因转录起始位点前1050bp到转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
NNMT基因转录起始位点前1050bp到转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
NNMT基因转录起始位点前840bp到转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
实施例1
实验背景：细胞所需氧气主要由线粒体氧化磷酸化通路所消耗，对线粒体氧消耗速率(OCR)的分析可直接反映线粒体氧化磷酸化通路的活性。本实施例采用Seahorse XFe代谢分析仪检测NCI-H82细胞在线粒体氧化磷酸化通路抑制剂Oligomycin A、Gboxin、S-Gboxin和IACS-010759存在以及不存在情况下其氧气消耗情况。
实验方法和结果：将NCI-H82(源自ATCC，编号HTB-175)细胞培养于添加10％FBS(加P/S)RPMI1640培养基，分别加入线粒体氧化磷酸化通路抑制剂Oligomycin A(1μM)、Gboxin(2μM)、S-Gboxin(5μM)、IACS-010759(1μM)，并设有不加任何线粒体氧化磷酸化通路抑制剂的对照组，在0.5小时内完成对细胞氧消耗的检测，实验结果如图1所示。
从图1中可以看出，相比不加任何线粒体氧化磷酸化抑制剂的对照组，在NCI-H82细胞中加入小分子化合物Oligomycin A、Gboxin、S-Gboxin以及IACS-010759可显著抑制其氧消耗，表明这些小分子可有效抑制线粒体氧化磷酸化通路。
实施例2
随机选取来源于不同组织并且基因型各异的细胞系，使用细胞活性检测试剂检测其对线粒体氧化磷酸化通路抑制剂Gboxin化合物的敏感性，结果发现一部分细胞株对Gboxin化合物敏感，IC 50值较低，而另有一部分细胞株对Gboxin化合物不敏感，IC 50值较高。
实验背景：细胞活力检测采用Promega CellTiter-Glo试剂盒，该试剂盒通过直接检测细胞内ATP含量反映所测细胞的活力。本实验检测线粒体氧化磷酸化通路抑制剂Gboxin化合物对不同肿瘤细胞系细胞活力抑制的IC 50值，各肿瘤细胞系名称、来源及培养条件如下：
细胞系NCI-H82(ATCC,编号HTB-175)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系G-401(ATCC，编号CRL-1441)培养于含10％胎牛血清的McCoy's 5a培养基+P/S；
细胞系MDA-MB-453(ATCC，编号HTB-131)培养于含10％胎牛血清的Leibovitz's L-15培养基+P/S；
细胞系WSU-DLCL2(DSMZ，编号ACC-575)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系SU-DHL-2(ATCC，编号CRL-2956)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系OCI-AML-3(DSMZ，编号ACC-582)培养于含20％胎牛血清的RPMI1640培养基+P/S；
细胞系SW48(ATCC，编号CCL-231)培养于含10％胎牛血清的Leibovitz's L-15培养基+P/S；
细胞系ATN-1(RIKEN，编号RBRC-RCB1440)培养于含10％胎牛血清及0.1mM NEAA的RPMI1640培养基+P/S；
细胞系HCC15(KCLB，编号70015)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系OCI-LY-19(DSMZ，编号ACC-528)培养于含10-20％h.i.FBS的80-90％alpha-MEM培养基+P/S；
细胞系22RV1(ATCC，编号CRL-2505)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系MIA PaCa-2(ATCC，编号CRL-1420)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系CCRF-CEM(ATCC，编号CCL-119)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系HH(ATCC，编号CRL-2105)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系OCI-AML-5(DSMZ，编号ACC-247)培养于alpha-MEM培养基(含20％胎牛血清及10％体积分数的经5637细胞系调整的培养基)+P/S；
细胞系G-402(ATCC，编号CRL-1440)培养于含10％胎牛血清的McCoy's 5a培养基+P/S；
细胞系HCC1806(ATCC,编号CRL-2335)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系BT-549(ATCC,编号HTB-122)培养于含10％胎牛血清0.023IU/ml人胰岛素的RPMI1640培养基+P/S；
细胞系OCI-AML-4(DSMZ，编号ACC-729)培养于alpha-MEM培养基(含20％胎牛血清及20％体积分数的经5637细胞系调整的培养基)+P/S；
细胞系H9(ATCC，编号HTB-176)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系Jurkat,Clone E6-1(ATCC，编号TIB-152)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系G-361(ATCC，编号CRL-1424)培养于含10％胎牛血清的McCoy's 5a培养基+P/S。
细胞系U-937(ATCC，编号CRL-1593.2)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系SNU-398(ATCC，编号CRL-2233)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系NCI-H1048(ATCC，编号CRL-5853)培养于含5％胎牛血清的HITES培养基+P/S；
细胞系A-375(ATCC，编号CRL-1619)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系D283 Med(ATCC，编号HTB-185)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系GAK(JCRB，编号JCRB0180)培养于含20％胎牛血清的Ham's F12培养基+P/S；
细胞系CHL-1(ATCC，编号CRL-9446)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系NCI-H1155(ATCC，编号CRL-5818)培养于无血清的ACL-4培养基+P/S；
细胞系LS 180(ATCC，编号CL-187)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系Daoy(ATCC,编号HTB-186)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系DU 145(ATCC，编号HTB-81)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系AM-38(JCRB，编号IFO50492)培养于含20％热灭活胎牛血清的EMEM培养基+P/S；
细胞系HCC70(ATCC，编号CRL-2315)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系PANC-1(ATCC，编号CRL-1469)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系U-87 MG(ATCC，编号HTB-14)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系MJ(ATCC,编号CRL-8294)培养于含20％胎牛血清的IMDM培养基+P/S；
细胞系Gp2D(ECACC,编号95090714)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系SU.86.86(ATCC,编号CRL-1837)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系NCI-H2081(ATCC,编号CRL-5920)培养于含5％胎牛血清的HITES培养基+P/S；
细胞系NCI-H1793(ATCC,编号CRL-5896)培养于含5％胎牛血清的HITES培养基+P/S；
细胞系ACHN(ATCC，编号CRL-1611)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系U-251 MG(ECACC,编号9063001)培养于含2mMGlutamine、1％NEAA、1mM Sodium Pyruvate(NaP)以及10％胎牛血清的EMEM培养基+P/S；
细胞系MDA-MB-231(ATCC,编号HTB-26)培养于含10％胎牛血清的Leibovitz's L-15培养基+P/S；
细胞系NCI-H196(ATCC,编号CRL-5823)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系PC-3(ATCC,编号CRL-1435)培养于含10％胎牛血清的F-12K培养基+P/S；
细胞系OCI-M1(DSMZ,编号ACC-529)培养于含20％胎牛血清的IMDM培养基+P/S；
细胞系NCI-H1651(ATCC,编号CRL-5884)培养于含10％胎牛血清的ACL-4培养基+P/S；
细胞系C3A(ATCC，编号CRL-10741)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系SNU-449(ATCC,编号CRL-2234)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系GB-1(JCRB,编号IFO50489)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系769-P(ATCC,编号CRL-1933)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系COLO 320HSR(ATCC,编号CCL-220.1)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系CFPAC-1(ATCC,编号CRL-1918)培养于含10％胎牛血清的IMDM培养基+P/S；
细胞系SF126(JCRB,编号IFO50286)培养于含10％胎牛血清的EMEM培养基+P/S；
细胞系786-O(ATCC,编号CRL-1932)培养于含10％胎牛血清的RPMI1640培养基+P/S；
实验方法和结果：上述各肿瘤细胞培养于相关培养基(加P/S)，传代3小时后加入梯度稀释的Gboxin化合物，培养3-4天后测定相关半抑制浓度IC 50。
实验结果如下表3所示。
表3 Gboxin化合物对不同肿瘤细胞株的IC 50(μM)
  IC50(μM)
NCI-H82 0.398
G-401 0.777
MDA-MB-453 0.942
WSU-DLCL2 0.682
SU-DHL-2 0.97
OCI-AML-3 1.46
SW48 1.535
ATN-1 1.55
HCC15 2.26
OCI-LY-19 2.51
22RV1 2.53
MIA PaCa-2 2.57
CCRF-CEM 2.59
HH 2.67
OCI-AML-5 2.74
G-402 2.82
HCC1806 3.18
BT-549 3.28
OCI-AML-4 3.34
H9 3.34
Jurkat,Clone E6-1 3.51
G-361 3.88
U-937 4.06
SNU-398 4.17
NCI-H1048 4.34
A-375 4.65
D283 Med 4.81
GAK 7.05
CHL-1 7.68
NCI-H1155 8.84
LS 180 8.90
Daoy 9.72
DU 145 9.91
AM-38 10.29
HCC70 11.23
PANC-1 11.99
U-87 MG 12.31
MJ 12.90
Gp2D 13.30
SU.86.86 13.68
NCI-H2081 13.92
NCI-H1793 15.99
ACHN 16.57
U-251 MG 16.68
MDA-MB-231 17.81
NCI-H196 19.31
PC-3 21.26
OCI-M1 21.65
NCI-H1651 22.36
C3A 25.74
SNU-449 >30.00
GB-1 >30.00
769-P >30.00
COLO 320HSR >30.00
CFPAC-1 >30.00
SF126 >30.00
786-O >30.00
备注：IC 50为半抑制浓度(50％inhibiting concentration)，即达到50％抑制效果时抑制剂的浓度。
由表3可知，对不同细胞进行线粒体氧化磷酸化通路抑制剂Gboxin敏感性检测发现NCI-H82(人小细胞肺癌细胞)、G-401(人肾癌Wilms细胞)、MDA-MB-453(乳腺癌细胞)、WSU-DLCL2(人弥漫大B淋巴瘤细胞)和SW48(人结肠腺癌细胞)等对Gboxin敏感(IC 50值较低)；而GB-1(人脑胶质母细胞瘤细胞)、CFPAC-1(人胰腺癌细胞)、SF126(人脑瘤细胞)、786-O(肾透明细胞腺癌细胞系)等对Gboxin不敏感(IC 50值较高)。
实施例3
使用细胞活性检测试剂进一步检验实施例2中发现的对线粒体氧化磷酸化通路抑制剂Gboxin小分子敏感的肿瘤细胞株(NCI-H82、G-401、MDA-MB-453、WSU-DLCL2、SW48)和不敏感肿瘤细胞株(GB-1、CFPAC-1、SF126、786-O)对线粒体氧化磷酸化通路抑制剂Oligomycin A、IACS-010759的敏感性。
实验背景：细胞活力检测采用Promega CellTiter-Glo试剂盒，该试剂盒通过直接检测细胞内ATP含量反应细胞活力。本实验检测线粒体氧化磷酸化通路抑制剂小分子Oligomycin A和IACS-010759对不同肿瘤细胞活力抑制的IC 50值。
实验方法和结果：各肿瘤细胞均培养于实施例2所述相关培养基中，细胞传代3小时后，分别加入梯度稀释的Oligomycin A和IACS-010759，培养3-4天后测定这两个线粒体氧化磷酸化通路抑制剂各自对相关细胞的半抑制剂量IC 50。
实验结果如表4所示：
表4化合物Oligomycin A和IACS-010759对不同细胞株的抑制作用(IC 50)
Figure PCTCN2021121088-appb-000041
Figure PCTCN2021121088-appb-000042
备注：IC 50为半抑制浓度(50％inhibiting concentration)，即达到50％抑制效果时抑制剂的浓度。
由表4可知，对线粒体氧化磷酸化通路抑制剂Gboxin敏感的细胞株(NCI-H82、G-401、MDA-MB-453、WSU-DLCL2、SW48)同样对线粒体氧化磷酸化通路抑制剂Oligomycin A、IACS-010759敏感(IC 50值相对较低)，而对Gboxin不敏感的细胞株(GB-1、CFPAC-1、SF126、786-O)同样对Oligomycin A、IACS-010759小分子不敏感(IC 50值相对较高)。
实施例4
用Western Blot实验检测对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82，G-401和WSU-DLCL2)在Gboxin和Oligomycin A等线粒体氧化磷酸化通路抑制剂小分子作用后，ATF4(转录激活因子4，Activating Transcription Factor 4)和mTOR(雷帕霉素靶蛋白)通路的活性变化情况。
实验背景：对线粒体氧化磷酸化通路的抑制会导致ATF4应激通路的激活和mTOR通路活性的下降，mTOR通路活性由磷酸化核糖体蛋白S6(即p-S6)表达量反映。ATF4的表达增加表明ATF4应激通路的激活，而p-S6蛋白表达降低表明mTOR通路活性受到抑制。
实验方法和结果：NCI-H82、G-401和WSU-DLCL2肿瘤细胞培养于含有10％FBS的相关培养基中(加p/s)，细胞传代过夜后，加入如图2所示1μM和3μM的Gboxin以及1μM的Oligomycin A，处理12小时后用Western Blot实验检测ATF4和p-S6蛋白的蛋白含量，实验结果如图2所示。
由图2可知，细胞株NCI-H82、G-401和WSU-DLCL2在Gboxin和Oligomycin A等线粒体氧化磷酸化通路抑制剂小分子作用后，ATF4应激通路上调，而p-S6蛋白降低说明mTOR通路活性受到抑制，表明这些细胞中氧化磷酸化通路活跃，对氧化磷酸化通路抑制剂敏感。
实施例5
用Western Blot实验检测对线粒体氧化磷酸化通路抑制剂不敏感的细胞(SF126、CFPAC-1和786-O)在Gboxin和Oligomycin A等线粒体氧化磷酸化通路抑制小分子作用后，ATF4和mTOR通路的活性变化情况。
实验背景：对线粒体氧化磷酸化通路的抑制会导致ATF4应激通路的激活和mTOR通路活性的下降，mTOR通路活性由磷酸化核糖体蛋白S6即p-S6表达量量反映。ATF4的表达增加表明ATF4应激通路的激活，而p-s6蛋白表达降低表明mTOR通路活性受到抑制。
实验方法和结果：SF126、CFPAC-1和786-O细胞培养于含有10％FBS的相关培养基中(加p/s)，细胞传代过夜后，加入如图3所示1μM和3μM的Gboxin以及1μM的 Oligomycin A，处理12小时后Western Blot实验检测ATF4和p-S6蛋白的蛋白含量，实验结果如图3所示。
由图3可知，细胞株SF126、CFPAC-1和786-O在Gboxin和Oligomycin A等线粒体氧化磷酸化通路抑制剂小分子作用后，ATF4应激通路和mTOR通路活性无明显变化，对氧化磷酸化通路抑制剂不敏感。
实施例6
利用生物信息学的方法检测对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)基因转录表达情况。
实验背景：细胞的行为和特性是由该细胞所表达的基因决定的，通过全基因组基因转录测序可以精确获得某细胞各个基因mRNA的转录水平，对所有基因mRNA的转录水平进行生物信息学计算分析可以将不同细胞按基因表达的近似程度进行分类。
实验方法和结果：对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)进行全基因组mRNA转录水平进行测序，然后利用生物信息学的方法对各细胞mRNA转录组的相似性进行计算分析。
实验结果如图4所示,图中pc是指principal component，pc1表示综合各基因表达信息显示的细胞间差异程度。
由图4可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)在基因转录水平上是明显不同的两类细胞。
实施例7
利用生物信息学的方法分析对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)差异表达基因的功能情况。
实验背景：细胞的行为和特性是由该细胞所表达的基因决定的，多个(或多组)细胞间差异表达基因往往决定了这些细胞的不同特性。对多个(或多组)细胞差异表达基因mRNA转录水平进行生物信息学计算分析可以获得这些细胞具有的不同特性。
实验方法和结果：利用生物信息学的方法分析得到对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)之间存在的差异表达基因，然后对这些差异表达基因进行功能分析以获得这两组细胞在功能方面的区别，实验结果如图5所示。
由图5可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1、GB-1、SF126)之间的差异表达上调基因主要集中在与代谢相关的通路上(如碳代谢、丙酮酸代谢、丙酸代谢、乙醛酸和二羧酸代谢等)，表明这两群细胞在细胞代谢方面存在明显的差异，敏感细胞中相关代谢通路上调。
实施例8
利用生物信息学的方法分析对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)在代谢通路方面的主要差异。
实验背景：由实施例7可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1、GB-1、SF126)在与代谢相关的通路上存在显著差异。细胞代谢是由多条代谢通路的活性(基因表达)决定的，深入分析这两组细胞间不同的代谢通路可更深入地知悉这两类细胞在代谢方面的不同之处。
实验方法和结果：利用生物信息学的方法分析得到对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的肿瘤细胞株(786-O、CFPAC-1、GB-1、SF126)之间的差异表达基因，选取其中参与细胞代谢的差异基因分析这些基因所参与的代谢通路，实验结果如图6所示。
由图6可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1、GB-1、SF126)之间差异表达最显著的代谢通路是线粒体氧化磷酸化通路(oxidative phosphorylation)，其在敏感细胞中显著上调。
实施例9
利用生物信息学的方法分析对线粒体氧化磷酸化通路抑制剂敏感的肿瘤细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的肿瘤细胞株(786-O、CFPAC-1、GB-1、SF126)在氧化磷酸化通路蛋白复合体方面的差异。
实验背景：据实施例8可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)在线粒体氧化磷酸化通路上存在显著差异，氧化磷酸化通路是由包含90多个蛋白的5个蛋白复合体组成。
实验方法和结果：利用生物信息学的方法分析得到对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)之间的差异表达基因，选取与氧化磷酸化通路蛋白复合体有关的差异表达基因并分析得到这些基因所参与表达的氧化磷酸化通路蛋白复合体，实验结果如图7所示。
由图7可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1、SF126)在氧化磷酸化通路上的差异表达基因主要集中于氧化磷酸化通路蛋白复合体I,III,IV,和V，这些蛋白在敏感细胞中高表达。
实施例10
利用线粒体膜电差指示剂分析对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1和SF126)之间在膜电差上的可能差异。
实验背景：线粒体膜电差可调控线粒体蛋白转运、自噬、ATP合成等多种重要线粒体功能，对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1和SF126)在氧化磷酸化通路上存在显著差异，这些差异也会反映在线粒体膜电势差上。
实验方法和结果：使用线粒体膜电差指示剂TMRE(tetramethylrhodamine,ethyl ester)检测正常培养状态下对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1和SF126)的膜电差，实验结果如图8所示。
由图8可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401和WSU-DLCL2)膜电差相对较高，不敏感的细胞(786-O、CFPAC-1和SF126)线粒体膜电差相对较低，两组细胞在线粒体膜电差上存在显著差异。
实施例11
利用Seahorse细胞代谢分析仪分析对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1和SF126)在氧气消耗方面的差异。
实验背景：细胞所需90％以上的氧气是由线粒体氧化磷酸化通路消耗的，因此细胞氧化磷酸化通路的活性和其氧气消耗水平紧密相关。对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1和SF126)在氧化磷酸化通路蛋白表达上存在显著差异，相关蛋白在敏感细胞上是高表达的，这些差异也会反映在以氧气消耗上。
实验方法和结果：按照Seahorse细胞代谢分析仪的标准流程检测正常培养状态下对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1和SF126)的线粒体氧消耗速率(OCR)。实验结果如图9所示。
由图9可知，对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401和WSU-DLCL2)有显著高于不敏感细胞株(786-O、CFPAC-1和SF126)的氧气消耗水平。
实施例12
利用转录组和生物信息学的方法筛选对线粒体氧化磷酸化通路抑制剂敏感的肿瘤细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的肿瘤细胞株(786-O、CFPAC-1、GB-1、SF126)之间表达差异显著的基因。
实验背景：精准用药是现代药物研发的方向，生物标记物为实现精准用药提供了极好的技术保障，一个好的生物标记物应具有能明确区分药物应答组和非应答组的能力，大部分生物标记物是某个基因或某组基因的表达。
实验方法和结果：通过对线粒体通路抑制剂敏感的细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1、GB-1、SF126、CFPAC)进行转录组测序，用生物信息学的方法筛选在这两组细胞中表达差异显著的基因，实验结果如图10所示。
从图10中可以看出，对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞(786-O、CFPAC-1、GB-1、SF126) 在NNMT基因表达方面有非常大的差异，氧化磷酸化通路抑制剂敏感的细胞中NNMT基因是低表达的。
实施例13
在细胞水平检测NNMT基因转录水平作为对线粒体氧化磷酸化通路抑制剂敏感性生物标记物的可行性。
实验背景：精准用药是现代药物研发的方向。生物标记物为实现精准用药提供了极好的技术保障。一个好的生物标记物应具有能明确区分药物应答组和非应答组的能力，并且和所对应的生物特征成正比例或者反比例关系。
实验方法和结果：根据实施例2中获得的来自不同组织来源且基因型各异的57株肿瘤细胞对线粒体氧化磷酸化通路抑制剂Gboxin的敏感性数据，按照IC 50数值高低不同分成五组，即组一：IC 50<1μM；组二：1μM<IC 50<3μM；组三：3μM<IC 50<9μM；组四：9μM<IC 50<27μM；组五：27μM<IC 50。测定每组所有肿瘤细胞的平均NNMT基因转录水平，分析肿瘤细胞NNMT基因转录水平和氧化磷酸化抑制剂敏感性的相关性。氧化磷酸化抑制剂Gboxin对肿瘤细胞的抑制效果(IC 50)与NNMT基因转录水平的关系如图11所示。
从图11中可以看出各细胞NNMT基因转录水平和其对Gboxin的敏感性(IC 50越小则越敏感)呈指数负相关，表明NNMT基因转录水平和肿瘤细胞对线粒体氧化磷酸化通路抑制剂的敏感性呈负相关，即细胞NNMT基因转录水平越低则该肿瘤细胞对线粒体氧化磷酸化通路抑制剂的敏感性越高。
实施例14
对线粒体氧化磷酸化通路抑制剂敏感的细胞和不敏感的细胞在mRNA和蛋白表达水平方面对NNMT基因进行验证。
实验背景：细胞中的基因通常是在蛋白水平行使其功能的，本实验检测NNMT基因的mRNA和蛋白水平。
实验方法和结果：利用RT-qPCR和western blot实验在对线粒体氧化磷酸化通路抑制剂敏感的细胞和不敏感的肿瘤细胞株中检测NNMT的mRNA和NNMT蛋白。实验结果如图12所示。
从图12中可以看出，NNMT基因的mRNA和蛋白在对线粒体氧化磷酸化通路抑制剂敏感的细胞(NCI-H82、G-401、SW48和WSU-DLCL2)中是低表达的，而在对线粒体氧化磷酸化通路抑制剂不敏感的细胞(786-O、CFPAC-1和SF126)中是高表达的。
实施例15
实验方法和结果：通过对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的细胞株(786-O、CFPAC-1、GB-1和SF126)进行全基因组转录组测序和DNA甲基化测序，用生物信息学的方法找出相对于不敏感肿瘤细胞而言敏感细胞株中基因低表达但其启动子区高甲基化的基因(红色圆点，Ddown-CpG_hyper，图13中左上部分)和敏感细胞株中高表达但其启动子区低甲基化的基因(深蓝色圆点，DEG_up-CpG_hypo，图13中右下部分)。
实验结果如图13所示，图中X轴表示某一基因转录表达水平在敏感细胞和不敏感细 胞中的比值(敏感细胞/不敏感细胞)，Y轴表示某一基因启动子区CpG岛甲基化水平在敏感细胞和不敏感细胞中的比值(敏感细胞/不敏感细胞)。
从图13中可以看出，NNMT基因启动子区在对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)中是高甲基化、低表达的，而在不敏感的肿瘤细胞株(786-O、CFPAC-1、GB-1和SF126)是低甲基化、高表达的。
实施例16
对Oligomycin A、Gboxin等线粒体氧化磷酸化通路抑制剂敏感的5例肿瘤细胞系(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的4例肿瘤细胞系(786-O、CFPAC-1、GB-1和SF126)的NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域以及NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域进行重亚硫酸盐测序以检测相关区域内DNA CpG位点甲基化水平，首先利用重烟硫酸盐对基因组DNA进行处理，将未发生甲基化的胞嘧啶脱氨基变成尿嘧啶，而发生了甲基化的胞嘧啶未发生脱氨基，因而可以基于此将经重亚硫酸盐处理的和未处理的测序样本进行比较来发现甲基化的位点，结果如图14、图15和图16所示。
如图14(NNMT基因启动子区)、图15(NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域)及图16(NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域)所示，线粒体氧化磷酸化通路抑制剂对NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域、NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域内DNA CpG位点甲基化水平高的肿瘤细胞的抑制作用显著较强，对NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域、NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域内DNA CpG位点甲基化水平低的肿瘤细胞的抑制作用显著较弱，表明肿瘤细胞NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域、NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域内DNA CpG位点甲基化水平与其对线粒体氧化磷酸化通路抑制剂的敏感性呈正相关。
实施例17
对线粒体氧化磷酸化通路抑制剂敏感的4例肿瘤细胞系(NCI-H82、G-401、SW48和WSU-DLCL2)和不敏感的3例肿瘤细胞系(786-O、CFPAC-1和SF126)的NNMT基因转录起始位点前840bp(即人11号染色体第114165695位)到基因转录起始位点前469bp(即人11号染色体第114166066位)区域内特定DNA CpG位点甲基化情况进行研究。
首先对细胞基因组DNA进行重亚硫酸盐处理，将未发生甲基化的胞嘧啶脱氨基变成尿嘧啶，而发生了甲基化的胞嘧啶未发生脱氨基，因而可以基于此将经重亚硫酸盐处理的和未处理的测序样本进行比较来发现甲基化的位点，随后用相应引物对该区域进行PCR扩增、测序分析以检测该DNA区域内CpG位点的甲基化水平。
分析发现对线粒体氧化磷酸化通路抑制剂敏感的G-401、SW48、NCI-H82、WSU-DLCL2细胞系中该区域内7个CpG位点(人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位、114166066位)几乎全 部被甲基化，而对线粒体氧化磷酸化通路抑制剂不敏感的CFPAC-1、786-O、SF126细胞系中该区域这7个CpG位点都未被甲基化，相关位点甲基化情况如图17所示。
其中，人11号染色体114165695、114165730、114165769、114165804、114165938、114166050、114166066的位点对应于SEQ ID NO:1核苷酸序列的位点如下所示：
Figure PCTCN2021121088-appb-000043
实施例18
用酶联免疫的方法检测对线粒体氧化磷酸化通路抑制剂敏感和不敏感细胞株内甲基化供体S-腺苷甲硫氨酸(SAM)的水平。
实验方法和结果：通过酶联免疫的方法对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、SW48和WSU-DLCL2)和不敏感细胞株(786-O、CFPAC-1和SF126)的甲基化供体SAM水平进行检测。实验结果如图18所示：
从图18中可以看出，对线粒体氧化磷酸化通路抑制剂敏感的细胞株(NCI-H82、G-401、SW48和WSU-DLCL2)中甲基化供体SAM水平明显高于对线粒体氧化磷酸化通路抑制剂不敏感的细胞株(786-O、CFPAC-1和SF126)中甲基化供体SAM的水平。
实施例19
细胞DNA的甲基化水平由DNA甲基化酶DNMT3a、DNMT3b和DNMT1维持，DNMT3a、DNMT3b能对DNA进行从头进行甲基化，而DNMT1能在蛋白UHRF1(泛素样含PHD和环指域蛋白1)的帮助下对已甲基化的DNA进行复制维持，本实施例检测肿瘤中NNMT表达与DNMT1、UHRF1、DNMT3a以及DNMT3b表达的相关性。
实验方法和结果：从公共数据库中(Cancer Cell Line Encyclopedia，CCLE,共1019株细胞)得到多种细胞中NNMT基因、DNMT1、UHRF1、DNMT3a以及DNMT3b的表达数据，然后用生物信息学的方法分析这些细胞中NNMT表达和DNMT1、UHRF1、DNMT3a、DNMT3b表达的相关性，分析各细胞NNMT基因表达水平和DNMT1、UHRF1、DNMT3a以及DNMT3b的表达水平的相关性，实验结果如附图19所示：
从图19中可以看出，各细胞中NNMT的表达和DNA甲基化酶、UHRF1的表达呈负相关。
实施例20
在细胞水平考察DNMT1(DNA Methyltransferase1)基因转录水平作为对线粒体氧化磷酸化通路抑制剂敏感性的生物标记物。
实验方法和结果：根据实施例2中获得的来自不同组织来源且基因型各异的57株肿瘤细胞对线粒体氧化磷酸化通路抑制剂Gboxin的敏感性数据，按照IC 50数值高低不同分 成五组，即组一：IC 50<1μM；组二：1μM<IC 50<3μM；组三：3μM<IC 50<9μM；组四：9μM<IC 50<27μM；组五：27μM<IC 50。
测定每组内所有细胞平均DNMT1基因转录mRNA水平，分析各肿瘤细胞DNMT1基因转录水平和氧化磷酸化敏感性的相关性。实验结果如图20所示：
从图20中可以看出，DNMT1基因转录水平和这些细胞对Gboxin的敏感性(IC 50越小则越敏感)呈指数正相关，表明DNMT1基因转录水平和相关细胞对线粒体氧化磷酸化通路抑制剂的敏感性呈正相关，即肿瘤细胞DNMT1基因转录水平越高则其对线粒体氧化磷酸化通路抑制剂敏感性越高。
实施例21
相关细胞NNMT表达水平和其对氧化磷酸化通路抑制剂的敏感性呈显著负相关，而其DNMT1基因表达水平和其对氧化磷酸化通路抑制剂的敏感性呈显著正相关，可进一步检测NNMT和DNMT1在相关细胞对氧化磷酸化通路抑制剂敏感性中的作用。
实验方法和结果：通过病毒载体将NNMT基因导入到NCI-H82细胞中使得NCI-H82细胞过表达NNMT蛋白，通过转染shRNA的方法敲低NCI-H82细胞DNMT1的表达，用检测胞内ATP的方法检测过表达NNMT蛋白和/或敲低DNMT1的表达后，细胞对线粒体氧化磷酸化通路抑制剂(Gboxin、Oligomycin A)敏感性的变化，实验结果如图21和图22所示，Vector为转染有空病毒的NCI-H82细胞对照组。
从图21和图22看出单独过表达对氧化磷酸化通路抑制剂敏感的NCI-H82细胞的NNMT蛋白(图中ov-NNMT)或分别使用两种不同针对DNMT1基因的shRNA敲低NCI-H82细胞的DNMT1表达(图中sh-DNMT1#1为一种针对DNMT1基因的shRNA,其对应的DNA序列为：GATCCGGCCCAATGAGACTGACATCAATTCAAGAGATTGATGTCAGTCTCATTGGGCTTTTTG(SEQ ID No:2)，sh-DNMT1#2为另外一种针对DNMT1基因的shRNA，其对应的DNA序列为：GATCCGGGATGAGTCCATCAAGGAAGATTCAAGAGATCTTCCTTGATGGACTCATCCTTTTTTG)(SEQ ID No:3)，发现NCI-H82细胞对Gboxin、Oligomycin A等线粒体氧化磷酸化通路抑制剂的敏感性降低。当在NCI-H82细胞中过表达NNMT蛋白并且同时敲低DNMT1表达(图中ov-NNMT/sh-DNMT1#1和ov-NNMT/sh-DNMT1#2)，NCI-H82肿瘤细胞对Gboxin、Oligomycin A等线粒体抑制剂敏感性降低得更为显著。
其中，Western Blot实验检测相对于正常NCI-H82(Vector)相比，过表达NNMT蛋白的NCI-H82(ov-NNMT)的NNMT蛋白含量如图23所示。Western Blot实验检测相对于正常NCI-H82(shVector)相比，两种shRNA敲低肿瘤细胞的DNMT1表达的NCI-H82(sh-DNMT1#1或sh-DNMT1#2的-DNMT1)的DNMT1蛋白含量如图24所示。
因此，本实施例通过过表达NCI-H82细胞NNMT蛋白，以及敲低NCI-H82细胞DNMT1的表达，进一步证实肿瘤细胞的NNMT表达水平和其对氧化磷酸化通路抑制剂的敏感性呈显著负相关，而肿瘤细胞的DNMT1表达水平和其对氧化磷酸化通路抑制剂的敏感性呈显著正相关。
实施例22
为验证对线粒体氧化磷酸化通路抑制剂敏感的细胞在体内是否仍然维持对氧化磷酸 化通路抑制剂的敏感性，使用S-Gboxin对敏感细胞(NCI-H82)和不敏感细胞(CFPAC-1)接种的小鼠皮下肿瘤进行有效性检测。
实验方法和结果：将5x10 6个NCI-H82、NCI-H82-NNMT ov(通过病毒载体将NNMT基因导入到NCI-H82细胞中使得NCI-H82细胞过表达NNMT蛋白)和CFPAC-1肿瘤接种于裸鼠皮下，构建荷瘤小鼠，各组荷瘤小鼠腹腔注射线粒体氧化磷酸化通路抑制剂化合物S-Gboxin，给药剂量为10mg/kg/day，检测S-Gboxin对这些肿瘤的抑制情况，对照组以同样腹腔注射方式给溶媒，其中，肿瘤体积计算方法为：肿瘤体积＝1/2长x宽 2。实验结果如图25、图26和图27。
从图25、图26和图27中可以看出，化合物S-Gboxin可显著抑制敏感细胞NCI-H82接种的裸鼠皮下肿瘤，而对NCI-H82-NNMT ov接种的裸鼠皮下肿瘤抑制效果显著差于NCI-H82接种的裸鼠皮下肿瘤的抑制效果，并且S-Gboxin对不敏感细胞CFPAC-1接种的裸鼠皮下肿瘤无明显抑制效果，从而表明氧化磷酸化通路抑制剂对NNMT低表达肿瘤的抑制作用更强，即NNMT低表达的肿瘤对氧化磷酸化通路抑制剂的敏感性强，NNMT高表达肿瘤对氧化磷酸化通路抑制剂的敏感性较差。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种线粒体氧化磷酸化通路抑制剂的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤。
  2. 如权利要求1所述的用途，其特征在于，所述的肿瘤包括NNMT基因低表达或未表达的肿瘤；和/或
    所述肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
  3. 如权利要求1所述的用途，其特征在于，所述的肿瘤包括：
    线粒体氧化磷酸化通路上调的肿瘤；
    所述的肿瘤包括DNA甲基化酶高表达的肿瘤；
    所述的肿瘤包括UHRF1高表达的肿瘤；和/或
    所述的肿瘤包括NNMT基因核苷酸位点甲基化水平高的肿瘤。
  4. 如权利要求2所述的用途，其特征在于，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因启动子区DNA CpG位点甲基化水平。
  5. 如权利要求3所述的用途，其特征在于，所述DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述的肿瘤选自下组：肺癌、肾癌、乳腺癌、结肠癌、直肠癌、结直肠癌、淋巴癌、白血病、胰腺癌、脑瘤、肝癌、前列腺癌、黑色素癌，或其组合。
  7. 如权利要求1所述的用途，其特征在于，所述的线粒体氧化磷酸化通路抑制剂包括式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐；
    Figure PCTCN2021121088-appb-100001
    其中，
    R 1、R 2、R 3、R 4、R 6、R 7、R 8和R 9各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
    R 5为无、氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；其中，当R5为无时，
    Figure PCTCN2021121088-appb-100002
    为双键；当R5不为无时，
    Figure PCTCN2021121088-appb-100003
    为单键；或者当 R5不为无且
    Figure PCTCN2021121088-appb-100004
    为双键时，与R5相连的N原子为N +；
    Z 1为
    Figure PCTCN2021121088-appb-100005
    其中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基；
    所述的杂环烷基、杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子；
    或所述的线粒体氧化磷酸化通路抑制剂包括式II化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐；
    Figure PCTCN2021121088-appb-100006
    其中，
    R 25、R 26、R 27、R 28、R 29、R 30、R 31、R 32、R 33、R 34、R 35和R 36各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C1-C12卤代烷氧基、取代或未取代的C1-C12卤代烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
    Z 2和Z 3各自独立地为取代或未取代的C6-C12亚芳基、取代或未取代的3-12元亚杂芳基；
    n为0、1、2、3、4、5、6、7、8、9、10、11或12；
    所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基；
    所述的杂环烷基、杂芳基、亚芳基、亚杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子；
    或所述的线粒体氧化磷酸化通路抑制剂包括式III化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐；
    Figure PCTCN2021121088-appb-100007
    其中，
    R 48、R 49、R 50、R 51、R 52、R 53、R 54、R 55、R 56、R 57、R 58、R 59、R 60、R 61、R 62、R 63、R 64、R 65、R 66、R 67、R 68、R 69、R 70、R 71、R 72、R 73、R 74、R 75、R 76、R 77、R 78、R 79、R 80、R 81、R 82、R 83、R 84、R 85、R 86、R 87、R 88、R 89、R 90和R 91各自独立地为氢、卤素、羟基、巯基、氨基、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的3-12元杂环烷基、取代或未取代的C1-C12烷氧基、取代或未取代的C1-C12烷硫基、取代或未取代的C6-C12芳基、取代或未取代的5-12元杂芳基；
    所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基(如三氟甲基)、C3-C8卤代环烷基、卤素、硝基、-CN、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基、C1-C8卤代烷氧基、C1-C8卤代烷硫基、C6-C12芳基、5-10元杂芳基、甲磺酰基、磺酰基；
    所述的杂环烷基、杂芳基的杂环上各自独立地具有1-4个(优选为1、2、3个或4个)选自N、O和S的杂原子。
  8. 如权利要求1所述的用途，其特征在于，所述的线粒体氧化磷酸化通路抑制剂选自下组：
    Figure PCTCN2021121088-appb-100008
    Figure PCTCN2021121088-appb-100009
  9. 一种用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物，所述的标志物包括NNMT基因和/或NNMT基因区DNA CpG位点甲基化水平。
  10. 一种用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗的标志物，所述的标志物包括线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平。
  11. 一种检测试剂盒，其特征在于，所述的检测试剂盒包括：
    (i)用于检测NNMT基因表达和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂。
  12. 一种检测试剂盒，其特征在于，所述的检测试剂盒包括：
    (i)用于检测线粒体氧化磷酸化通路表达水平或活性、DNA甲基化酶表达水平、UHRF1表达水平和/或NNMT基因核苷酸位点甲基化水平的检测试剂。
  13. 如权利要求11或12所述的检测试剂盒的用途，其特征在于，用于制备一伴随诊断试剂盒，所述伴随诊断试剂盒用于判断肿瘤患者是否适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
  14. 一种药盒，其特征在于，所述的药盒包括：
    (i)用于检测NNMT基因表达和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂；和
    (ii)线粒体氧化磷酸化通路抑制剂。
  15. 一种药盒，其特征在于，所述的药盒包括：
    (i)用于检测线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平和/或NNMT基因核苷酸位点甲基化水平的检测试剂；和
    (ii)线粒体氧化磷酸化通路抑制剂。
  16. 一种预防和/或治疗肿瘤的方法，其特征在于，给所需的对象施用线粒体氧化磷酸化通路抑制剂。
  17. 一种装置或系统，所述的装置或系统包括：
    (i)检测模块，所述的检测模块用于检测线粒体氧化磷酸化通路表达水平或活性、NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平；
    (ii)输出模块，所述的输出模块包括输出以下信息：当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路上调、NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高 表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗；和/或
    当肿瘤患者的肿瘤细胞中线粒体氧化磷酸化通路下调、NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用线粒体氧化磷酸化通路抑制剂进行预防和/或治疗。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023174381A1 (zh) * 2022-03-16 2023-09-21 南京施江医药科技有限公司 一种含氮杂环类化合物及其用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115721655A (zh) * 2021-08-27 2023-03-03 南京施江医药科技有限公司 四环素类药物在治疗肿瘤中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106248946A (zh) * 2016-08-19 2016-12-21 浙江大学 一种酶联免疫检测试剂盒及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180044617A (ko) * 2016-10-24 2018-05-03 아주대학교산학협력단 Uhrf1 및 dnmt1을 포함하는 세포 노화 진단용 바이오마커 조성물
WO2018183668A1 (en) * 2017-03-30 2018-10-04 The Board Of Regents Of The University Of Texas System Quinoline derived small molecule inhibitors of nicotinamide n-methyltransferase (nnmt) and uses thereof
US20210386750A1 (en) * 2018-10-26 2021-12-16 Mayo Foundation For Medical Education And Research Methods and materials for treating cancer
CN114432307A (zh) * 2020-11-06 2022-05-06 南京施江医药科技有限公司 黄连碱类药物在治疗肿瘤中的应用
CN114644626A (zh) * 2020-12-18 2022-06-21 南京施江医药科技有限公司 一种苯环类化合物及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106248946A (zh) * 2016-08-19 2016-12-21 浙江大学 一种酶联免疫检测试剂盒及其应用

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Cancer Cell Line Encyclopedia"
"UniProt", Database accession no. P40261
"Uniprot", Database accession no. Q96T8 8
LIU YU'E, SHI YUFENG: "Mitochondria as a target in cancer treatment", MEDCOMM, vol. 1, no. 2, 1 September 2020 (2020-09-01), pages 129 - 139, XP055914469, ISSN: 2688-2663, DOI: 10.1002/mco2.16 *
MOLINA, J.R.SUN, YPROTOPOPOVA, M ET AL.: "An inhibitor of oxidative phosphorylation exploits cancer vulnerability", NAT MED, vol. 24, 2018, pages 1036 - 1046, XP036542082, DOI: 10.1038/s41591-018-0052-4
NATURE MEDICINE
RAMSDEN DAVID B., ROSEMARY H WARING, DAVID J BARLOW, RICHARD B PARSONS: "Nicotinamide N-Methyltransferase in Health and Cancer", INTERNATIONAL JOURNAL OF TRYPTOPHAN RESEARCH, vol. 10, 30 June 2017 (2017-06-30), pages 1 - 19, XP055914471, DOI: 10.1177/1178646917691739 *
See also references of EP4218752A4
SHI, YLIM, S.K.LIANG, Q ET AL.: "Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma", NATURE, vol. 567, 2019, pages 341 - 346, XP036735126, DOI: 10.1038/s41586-019-0993-x
VIALE, APETTAZZONI, PLYSSIOTIS, C ET AL.: "Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function", NATURE, vol. 514, 2014, pages 628 - 632, XP037437635, DOI: 10.1038/nature13611
ZHANG JUN, WANG YANZHONG, LI GUILING, YU HAITAO, XIE XINYOU: "Down-Regulation of Nicotinamide N-methyltransferase Induces Apoptosis in Human Breast Cancer Cells via the Mitochondria-Mediated Pathway", PLOS ONE, vol. 9, no. 2, 18 February 2014 (2014-02-18), pages e89202 - 13019, XP055914472, DOI: 10.1371/journal.pone.0089202 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023174381A1 (zh) * 2022-03-16 2023-09-21 南京施江医药科技有限公司 一种含氮杂环类化合物及其用途
WO2023174377A1 (zh) * 2022-03-16 2023-09-21 南京施江医药科技有限公司 一种四环类化合物及其用途

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