A nucleic acid molecule and inhibitor for inhibiting D2HGDH and its use

Abstract

The present invention relates to a nucleic acid molecule that specifically inhibits the transcription or translation of a D2HGDH gene or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene and uses thereof, and belongs to the fields of molecular biology and biomedicine technology. The nucleic acid molecule can specifically inhibit the transcription or translation of the D2HGDH gene, and the inhibitor can inhibit the activity of a protein encoded by the D2HGDH gene, and can be used to prepare a drug for treating tumors. The nucleic acid molecule has an excellent effect of specifically inhibiting the transcription or translation of the D2HGDH gene, and has excellent activity in treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and biomedicine technology, and in particular to a nucleic acid molecule that specifically inhibits the transcription or translation of a D2HGDH gene, an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene, and uses thereof. Background Art

[0002] The function of isocitrate dehydrogenase (IDH) is to catalyze the dehydrogenation of isocitrate to produce α-ketoglutarate (α-KG) and convert it into NAD + or NADP + Generate NADH or NADPH as a coenzyme. There are three subtypes of IDH in human cells, namely IDH1, IDH2 and IDH3. In 2008, a study published in Science magazine found that gliomas contain IDH1R132H mutations. A large number of subsequent studies have shown that this mutation mainly occurs in WHO grade II or III gliomas, with a mutation frequency of an astonishing 85-90%. In addition to gliomas, a large number of IDH1 mutations or IDH2 mutations have also been found in a variety of malignant tumors such as acute myeloid leukemia (10-20%), angioimmunoblastic T-cell lymphoma (10-40%), chondrosarcoma (56%), cholangiocarcinoma (20%) and sinus cancer (80%), indicating that these mutations are closely related to the occurrence of the corresponding tumors.

[0003] In 2009, Dang et al. reported a landmark discovery in Nature magazine that mutant IDH1 has acquired a new function and can reduce the wild-type IDH1 product α-KG to D-2-hydroxyglutarate (D-2HG). Subsequent studies have shown that tumor-related IDH1 mutations or IDH2 mutations have the common feature of producing D-2HG. D-2HG is extremely similar to α-KG in structure, so it can competitively inhibit the activity of various dioxygenases such as TET2 and KDMs that use α-KG as a substrate. TET2 and KDMs are important genes in the process of DNA and histone demethylation. D-2HG inhibits their function and leads to high methylation of DNA and histones, which reshapes the epigenetics of cells, affects the normal expression of many genes, and promotes the occurrence and development of tumors. Therefore, D-2HG is considered a "cancer metabolite."

[0004] It is worth noting that IDH1 mutation or IDH2 mutation is an early event in the process of tumorigenesis, and IDH1 mutation or IDH2 mutation exists in all cells inside the tumor. Therefore, IDH1 mutant or IDH2 mutant is considered to be an ideal therapeutic target. In recent years, pharmaceutical companies such as Agios have developed a variety of IDH1 mutant or IDH2 mutant inhibitors, such as Ivosidenib, Enasidenib, Vorasidenib and BAY1436032. At present, these IDH1 mutant or IDH2 mutant inhibitors have been clinically used to treat a variety of IDH1 mutant or IDH2 mutant tumors. Among them, Ivosidenib and Enasidenib have been approved for marketing in the United States. The complete remission rate of these two IDH1 mutant or IDH2 mutant inhibitors for the treatment of refractory or relapsed acute myeloid leukemia containing IDH1 mutations is about 20%. However, the results of clinical trials targeting solid tumors with IDH1 mutation or IDH2 mutation show that although IDH1 mutant or IDH2 mutant inhibitors can effectively inhibit the production of D-2HG, they have no obvious therapeutic effect on solid tumors including gliomas with IDH1 mutation or IDH2 mutation. Therefore, it is urgent to develop other treatment strategies for tumors with IDH1 mutation or IDH2 mutation. Summary of the invention SUMMARY OF THE INVENTION

[0006] In order to provide an effective treatment strategy for IDH1 mutation or IDH2 mutation tumors, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a use of a nucleic acid molecule that specifically inhibits the transcription or translation of a D2HGDH gene, or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene, in the preparation of a drug for treating a tumor. The present invention can effectively inhibit tumor growth by specifically inhibiting the transcription or translation of the D2HGDH gene or inhibiting the activity of a protein encoded by the D2HGDH gene.

[0008] In a second aspect, the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule is shRNA, siRNA and / or sgRNA.

[0009] In a third aspect, the present invention provides a recombinant vector, which comprises the nucleic acid molecule described in the second aspect and a viral vector.

[0010] In a fourth aspect, the present invention provides a nucleic acid molecule delivery system, wherein the nucleic acid molecule delivery system comprises the nucleic acid molecule described in the second aspect.

[0011] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0012] In a sixth aspect, the present invention provides a composition comprising the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the nucleic acid molecule delivery system described in the fourth aspect, the host cell described in the fifth aspect, and at least one of an antibody to a protein encoded by D2HGDH or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene.

[0013] In a seventh aspect, the present invention provides a method for delivering nucleic acid molecules to cells for non-therapeutic purposes. DETAILED DESCRIPTION OF THE INVENTION

[0015] To solve the aforementioned problems, the present invention provides the following technical solutions.

[0016] In a first aspect, the present invention provides a use.

[0017] A use of a nucleic acid molecule that specifically inhibits transcription or translation of a D2HGDH gene, or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene, in the preparation of a drug for treating a tumor, wherein the nucleic acid molecule is shRNA, siRNA and / or sgRNA.

[0018] The gene D2HGDH (Gene ID: 728294, Ensembl: ENSG00000180902) of the present invention is derived from humans and is located on human chromosome 2. Its mRNA contains 16 splice variants, among which the longest splice variant is NM_152783.5, and its nucleotide sequence is: Seq ID NO.46.

[0019] The D2HGDH protein, namely UniProtKB-Q8N465, has an amino acid sequence of: Seq ID NO.47.

[0020] In some embodiments, the target sequence of the nucleic acid molecule includes Seq ID NO.1, Seq ID NO.4, Seq ID NO.7, Seq ID NO.13, Seq ID NO.16 or Seq ID NO.19.

[0021] In some embodiments, the shRNA comprises shRNA-1, shRNA-2, or shRNA-3;

[0022] The shRNA-1 includes a sense strand and an antisense strand. The sense strand of the shRNA-1 contains Seq ID NO.2 (Seq ID NO.2: 5′-TGGAAGGACAACACGGGCTATTCAAGAGATAGCCCGTGTTGTCCTTCCTTTTTTC-3′), and the antisense strand of the shRNA-1 contains Seq ID NO.3 (Seq ID NO.3:

[0023] 5′-TCGAGAAAAAAGGAAGGACAACACGGGCTATCTCTTGAATAGCCCGTGTTGTCCTTCCA-3′) (the target sequence of the shRNA-1 is Seq ID NO. 1: 5′-GGAAGGACAACACGGGCTA-3′);

[0024] The shRNA-2 includes a sense strand and an antisense strand. The sense strand of the shRNA-2 contains Seq ID NO.5 (Seq ID NO.5: 5′-TGGGGAAACGTGGCAACCAATTCAAGAGATTGGTTGCCACGTTTCCCCTTTTTTC-3′), and the antisense strand of the shRNA-2 contains Seq ID NO.6 (Seq ID NO.6:

[0025] 5′-TCGAGAAAAAAGGGGAAACGTGGCAACCAATCTCTTGAATTGGTTGCCACGTTTCCCC-3′) (the target sequence of the shRNA-2 is Seq ID NO.4: 5′-GGGGAAACGTGGCAACCAA-3′);

[0026] The shRNA-3 comprises a sense strand and an antisense strand, wherein the sense strand of the shRNA-3 contains Seq ID NO.8 (Seq ID NO.8: 5′-TGGGCTGTGAACGTGGCTTTTTCAAGAGAAAAGCCACGTTCACAGCCCTTTTTTC-3′), and the antisense strand of the shRNA-3 contains Seq ID NO.9 (Seq ID NO.9:

[0027] 5′-TCGAGAAAAAAGGGCTGTGAACGTGGCTTTTCTCTTGAAAAAGCCACGTTCACAGCCCA-3′) (the target sequence of the shRNA-3 is Seq ID NO.7: 5′-GGGCTGTGAACGTGGCTTT-3′).

[0028] In some embodiments, the siRNA comprises siRNA-1, siRNA-2, or siRNA-3;

[0029] The siRNA is an RNA sequence containing 19 bases, and the siRNA-1 sequence is Seq ID NO.10:

[0030] 5′-GGAAGGACAACACGGGCUA-3′; the siRNA-2 sequence is Seq ID NO.11:

[0031] 5′-GGGGAAACGUGGCAACCAA-3′; the siRNA-3 sequence is Seq ID NO.12: 5′-GGGCUGUGAACGUGGCUUU-3′.

[0032] The siRNA-1, siRNA-2 and siRNA-3 sequences are the RNA forms of the target sequence of shRNA-1, the target sequence of shRNA-2 and the target sequence of shRNA-3 respectively, and their biological effects are equivalent to those of the corresponding shRNA.

[0033] In some embodiments, the sgRNA comprises sgRNA-1, sgRNA-2 or sgRNA-3;

[0034] The sgRNA-1 includes a sense strand and an antisense strand, the sense strand of the sgRNA-1 contains Seq ID NO.14 (Seq ID NO.14: 5′-CACCGACGGGACTTCATCATGCCGC-3′), and the antisense strand of the sgRNA-1 contains Seq ID NO.15 (Seq ID NO.15: 5′-AAACGCGGCATGATGAAGTCCCGTC-3′) (the target sequence of the sgRNA-1 is Seq ID NO.13: 5′-ACGGGACTTCATCATGCCGC-3′);

[0035] The sgRNA-2 includes a sense strand and an antisense strand, the sense strand of the sgRNA-2 contains Seq ID NO.17 (Seq ID NO.17: 5′-CACCGGAGAACGGCAAGCGCCGCAC-3′), and the antisense strand of the sgRNA-2 contains Seq ID NO.18 (Seq ID NO.18: 5′-AAACGTGCGGCGCTTGCCGTTCTCC-3′) (the target sequence of the sgRNA-2 is Seq ID NO.16: 5′-GAGAACGGCAAGCGCCGCAC-3′);

[0036] The sgRNA-3 includes a sense chain and an antisense chain, the sense chain of the sgRNA-3 contains Seq ID NO.20 (Seq ID NO.20: 5′-CACCGGCCGGGCACGATGCGCTCAA-3′), and the antisense chain of the sgRNA-3 contains Seq ID NO.21 (Seq ID NO.21: 5′-AAACTTGAGCGCATCGTGCCCGGCC-3′) (the target sequence of the sgRNA-3 is Seq ID NO.19: 5′-GCCGGGCACGATGCGCTCAA-3′).

[0037] In some embodiments, the inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene includes but is not limited to at least one of ginkgolic acid (C15:1) and NSC87877.

[0038] In some embodiments, the tumor is selected from a tumor carrying an IDH1 mutation or an IDH2 mutation.

[0039] IDH1 mutation refers to a mutation in which the mutated IDH1 protein can produce D-2HG, including: IDH1 R132H, IDH1R132C, IDH1R132G, IDH1 R132L, IDH1 R132S, IDH1 R132Q, IDH1 R132V, IDH1 R132P, IDH1R100Q, etc.

[0040] IDH2 mutation refers to a mutation in which the mutated IDH2 protein can produce D-2HG, including: IDH2 R140Q, IDH2R140C, IDH2R172K, IDH2 R172S, IDH2 R172G, IDH2 R172M, IDH2 R172S, IDH2 R172T, IDH2R172W, etc.

[0041] The amino acid sequence of IDH1 is: Seq ID NO.48:

[0042] MSKKISGGSVVEMQGDEMTRIIWELIKEKLIFPYVELDLHSYDLGIENRDATNDQVTKDAAEAIKKHNVG

[0043] VKCATITPDEKRVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGRHAYGDQYRAT

[0044] DFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGMYNQDKSIEDFAHSSFQMALSKGWPLYLS

[0045] TKNTILKKYDGRFKDIFQEIYDKQYKSQFEAQKIWYEHRLIDDMVAQAMKSEGGFIWACKNYDGDVQSD

[0046] SVAQGYGSLGMMTSVLVCPDGKTVEAEAAHGTVTRHYRMYQKGQETSTNPIASIFAWTRGLAHRAKLDNNKELAFFANALEEVSIETIEAGFMTKDLAACIKGLPNVQRSDYLNTFEFMDKLGENLKIKLAQAKL。

[0047] The amino acid sequence of IDH2 is: Seq ID NO.49:

[0048] MAGYLRVVRSLCRASGSRPAWAPAALTAPTSQEQPRRHYADKRIKVAKPVVEMDGDEMTRIIWQFIKEK

[0049] LILPHVDIQLKYFDLGLPNRDQTDDQVTIDSALATQKYSVAVKCATITPDEARVEEFKLKKMWKSPNGTIR

[0050] NILGGTVFREPIICKNIPRLVPGWTKPITIGRHAHGDQYKATDFVADRAGTFKMVFTPKDGSGVKEWEVY

[0051] NFPAGGVGMGMYNTDESISGFAHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFD

[0052] KNKIWYEHRLIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGLMTSVLVCPDGKTIEAEAA

[0053] HGTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETVESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNLDRALGRQ。

[0054] Each mutation site of the IDH1 mutant is a mutation at the corresponding position in the above IDH1 amino acid sequence Seq ID NO.48. For example, the amino acid sequence of IDH1 R132H is: Seq ID NO.50:

[0055] MSKKISGGSVVEMQGDEMTRIIWELIKEKLIFPYVELDLHSYDLGIENRDATNDQVTKDAAEAIKKHNVG

[0056] VKCATITPDEKRVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRAT

[0057] DFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGMYNQDKSIEDFAHSSFQMALSKGWPLYLS

[0058] TKNTILKKYDGRFKDIFQEIYDKQYKSQFEAQKIWYEHRLIDMVAQAMKSEGGFIWACKNYDGDVQSD

[0059] SVAQGYGSLGMMTSVLVCPDGKTVEAEAAHGTVTRHYRMYQKGQETSTNPIASIFAWTRGLAHRAKLDNNKELAFFANALEEVSIETIEAGFMTKDLAACIKGLPNVQRSDYLNTFEFMDKLGENLKIKLAQAKL. The mutation patterns of other IDH1 mutations (IDH1 R132C, IDH1 R132G, IDH1 R132L, IDH1 R132S, IDH1 R132Q, IDH1 R132V, IDH1R132P, IDH1 R100Q, etc.) are similar.

[0060] Each mutation site of the IDH2 mutant is a mutation at the corresponding position in the above IDH2 amino acid sequence Seq ID NO.49. For example, the amino acid sequence of IDH2 R140Q is: Seq ID NO.51:

[0061] MAGYLRVVRSLCRASGSRPAWAPAALTAPTSQEQPRRHYADKRIKVAKPVVEMDGDEMTRIIWQFIKEK

[0062] LILPHVDIQLKYFDLGLPNRDQTDDQVTIDSALATQKYSVAVKCATITPDEARVEEFKLKKMWKSPNGTIQ

[0063] NILGGTVFREPIICKNIPRLVPGWTKPITIGRHAHGDQYKATDFVADRAGTFKMVFTPKDGSGVKEWEVY

[0064] NFPAGGVGMGMYNTDESISGFAHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFD

[0065] KNKIWYEHRLIDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGLMTSVLVCPDGKTIEAEAA

[0066] HGTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETVESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNLDRALGRQ. The mutation patterns of other IDH2 mutations (IDH2 R140C, IDH2 R172K, IDH2R172S, IDH2 R172G, IDH2 R172M, IDH2 R172S, IDH2 R172T, IDH2 R172W, etc.) are similar.

[0067] In some embodiments, the tumor comprises glioma, acute myeloid leukemia, angioimmunoblastic T-cell lymphoma, chondrosarcoma, cholangiocarcinoma, or sinonasal cancer.

[0068] In the present invention, the structure of the Ginkgolic acid (C15:1) is:

[0069] In the present invention, the structure of NSC87877 is:

[0070] In a second aspect, the present invention provides a nucleic acid molecule.

[0071] A nucleic acid molecule, as shown in the nucleic acid molecule of the first aspect.

[0072] In a third aspect, the present invention provides a recombinant vector.

[0073] A recombinant vector, comprising the nucleic acid molecule described in the second aspect and a viral vector.

[0074] In some embodiments, the viral vector comprises a retroviral vector, a lentiviral vector, or an adenoviral vector.

[0075] In a fourth aspect, the present invention provides a nucleic acid molecule delivery system.

[0076] A nucleic acid molecule delivery system, comprising the nucleic acid molecule described in the second aspect.

[0077] In some embodiments, the nucleic acid molecule delivery system includes at least one of N-acetylated galactosamine coupling modification, lipid nanoparticles, polymer nanoparticles (such as polyethyleneimine and chitosan) and exosomes.

[0078] In a fifth aspect, the present invention provides a host cell.

[0079] A host cell, comprising the nucleic acid molecule of the second aspect or the recombinant vector of the third aspect.

[0080] In a sixth aspect, the present invention provides a composition.

[0081] A composition comprising the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the nucleic acid molecule delivery system described in the fourth aspect, the host cell described in the fifth aspect, and at least one of an antibody to a protein encoded by D2HGDH or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene.

[0082] In some embodiments, the inhibitor includes at least one of ginkgolic acid C15:1 (CAS No. of ginkgolic acid C15:1 is 22910-60-7) and NSC87877 (CAS No. of NSC87877 is 56932-43-5).

[0083] In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant.

[0084] In some embodiments, the pharmaceutically acceptable adjuvant includes dimethyldioctadecyl ammonium (DDA) and / or monophosphoryl lipid A (MPL) and / or saline or other adjuvants.

[0085] In some embodiments, the other adjuvant comprises aluminum adjuvant or Freund's adjuvant.

[0086] In some embodiments, the composition can be in various forms such as injection, tablet, capsule, powder, paste, nanoformulation, etc. The above-mentioned various dosage forms of drugs can be prepared according to the methods in the field of gene drugs / oligonucleotide drugs.

[0087] In a seventh aspect, the present invention provides a method for delivering nucleic acid molecules to cells for non-therapeutic purposes.

[0088] A method for delivering a nucleic acid molecule to a cell for non-therapeutic purposes, comprising contacting the cell with at least one of the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the nucleic acid molecule delivery system described in the fourth aspect, the host cell described in the fifth aspect, or the composition described in the sixth aspect.

[0089] Beneficial Effects

[0090] Compared with the prior art, a certain embodiment of the present invention has at least one of the following beneficial technical effects:

[0091] (1) The present invention specifically inhibits the transcription or translation of the D2HGDH gene through nucleic acid molecules, or inhibits the activity of the protein encoded by the D2HGDH gene through an inhibitor (preferably at least one of ginkgolic acid C15:1 (the CAS number of ginkgolic acid C15:1 is 22910-60-7) and NSC87877 (the CAS number of NSC87877 is 56932-43-5)) or an antibody, which is beneficial for inhibiting the growth of tumors with IDH1 mutation or IDH2 mutation (the tumors include glioma, acute myeloid leukemia, angioimmunoblastic T-cell lymphoma, chondrosarcoma, cholangiocarcinoma or sinus cancer, etc.).

[0092] (2) The nucleic acid molecule of the present invention is shRNA, siRNA and / or sgRNA, wherein the shRNA is preferably selected from shRNA-1, shRNA-2 or shRNA-3; the siRNA is preferably selected from siRNA-1, siRNA-2 or siRNA-3; the sgRNA is preferably selected from sgRNA-1, sgRNA-2 or sgRNA-3, which can effectively inhibit the expression of D2HGDH protein in tumor cells.

[0093] (3) The inhibitor of the present invention is an inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene and can effectively inhibit the activity of the D2HGDH protein in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1The Western blot diagrams are for screening effective shRNA and sgRNA target sequences for knocking down or knocking out D2HGDH in Example 1. Figure A shows that six pairs of independent shRNAs were used to knock down D2HGDH expression in U251 cells, and the expression of D2HGDH was detected by Western blot; Figure B shows that six independent siRNAs were used to knock down D2HGDH expression in U251 cells, and the expression of D2HGDH was detected by Western blot; Figure C shows that six pairs of independent sgRNAs were used to knock down D2HGDH expression in U251 cells, and the expression of D2HGDH was detected by Western blot.

[0095] Figure 2 It is a diagram of the construction results of overexpressing IDH1 wild type or overexpressing IDH2 wild type and overexpressing IDH1 mutant or overexpressing IDH2 mutant cell lines in Example 2. Figure A shows the D-2HG concentration in the culture medium of U251 overexpressing IDH1 wild type cell line (Flag IDH1 WT) or overexpressing IDH1 mutant cell line (Flag IDH1 R132H) or U118 overexpressing IDH1 wild type cell line (FlagIDH1 WT) or overexpressing IDH1 mutant cell line (Flag IDH1 R132H) (bar graph in the figure), and Western blot detection of intracellular IDH1 (Flag) or IDH1 R132H and D2HGDH protein expression (Western blot graph in the figure). Figure B shows the D-2HG concentration in the culture medium of JURKAT cells overexpressing IDH2 WT or IDH2 R140Q (bar graph in the figure), and Western blot detection of intracellular IDH2 (Flag) or IDH2R140Q and D2HGDH protein expression (Western blot graph in the figure).

[0096] Figure 3 The expression of D2HGDH was knocked down in U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HT1080 and SW1353 cells using three pairs of independent shRNAs in Example 2, a cell proliferation curve graph drawn by cell counting (the curve graph in the figure), and a graph of D2HGDH protein expression in cells detected by Western blot (the Western blot graph in the figure).

[0097] Figure 4The figure is the result of the decreased proliferation ability of IDH1 mutant tumor or IDH2 mutant tumor cells caused by knocking down D2HGDH in Example 3. Figures A, B, C and D are the result of knocking down the expression of D2HGDH in U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HT1080 and SW1353 cells using shRNA, and complementing the wild-type D2HGDH (rWT) and the G477R mutant (rG477R) lacking D2HGDH enzyme activity; wherein the curve graph is a cell proliferation curve drawn by cell counting, and the Western blot graph shows the expression of D2HGDH protein in the cells.

[0098] Figure 5 The figure shows the result of knocking down D2HGDH to inhibit the tumorigenicity of IDH1 mutant tumor cells in vivo in Example 4. The expression of D2HGDH was knocked down in HT1080 cells, and the wild-type D2HGDH (rWT) and its enzyme-deficient G477R mutant (rG477R) were complemented, and these cells were injected subcutaneously into nude mice for tumorigenicity experiments; Figure A is a representative picture of tumor-bearing nude mice, Figure B is a statistical result of subcutaneous tumor volume, and Figure C is a statistical result of subcutaneous tumor weight.

[0099] Figure 6 The figure is a result diagram of the construction and optimization of the D2HGDH inhibitor screening system in Example 5. By adjusting the concentrations of D2HGDH protein, D-2HG, FAD+ and INT in the D2HGDH enzyme activity reaction system, the enzyme activity reaction meets the requirements of drug screening. Figure A shows the combination information of different component concentrations in the reaction system and the corresponding signal-to-noise ratio; Figure B shows the enzyme activity reaction curves of various reaction systems in Figure A.

[0100] Figure 7 The figure is a flow chart and result chart of high-throughput screening of D2HGDH small molecule inhibitors in Example 5. Figure A is a diagram of the high-throughput drug screening operation steps; Figure B is a graph showing the inhibition curve of D2HGDH enzyme activity by the screened D2HGDH candidate small molecule inhibitors; Figure C is the half-inhibitory concentration (IC50) of Ginkgolic acid (C15:1) and NSC87877 on D2HGDH.

[0101] Figure 8The results of the decreased proliferation ability of IDH1 mutant tumor or IDH2 mutant tumor cells caused by Ginkgolic acid (C15: 1) in Example 5 are shown in Figure A. Figure A is a statistical result of the cell proliferation rate after treating a series of gradient concentrations of Ginkgolic acid (C15: 1) (GA) in U251 IDH1 WT and U251 IDH1 R132H cells. Figures B, C, D and E are the experimental results corresponding to Figure A in U118 IDH1 WT and U118 IDH1R132H, JURKAT IDH2 WT and JURKAT IDH2 R140Q, HuCCT1IDH1 WT and HuCCT1 IDH1 R132H, HT1080, and SW1353 cells, respectively. Figure F is a statistical result of the half-inhibitory concentration of Ginkgolic acid (C15: 1) on the above-mentioned cell proliferation rate.

[0102] Fig. 9 The results of the decreased proliferation ability of IDH1 mutant tumors or IDH2 mutant tumor cells caused by NSC87877 in Example 5 are shown in Figure A. Figure A is a statistical result of the cell proliferation rate after treating a series of gradient concentrations of NSC87877 (NSC) in U251 IDH1 WT and U251 IDH1 R132H cells. Figures B, C, D and E are the experimental results corresponding to Figure A in U118 IDH1 WT and U118 IDH1 R132H, JURKAT IDH2 WT and JURKAT IDH2 R140Q, HuCCT1 IDH1 WT and HuCCT1 IDH1R132H, HT1080, and SW1353 cells, respectively. Figure F is a statistical result of the half-inhibitory concentration of NSC87877 on the above-mentioned cell proliferation rate.

[0103] Definition of terms:

[0104] In the present invention, "room temperature" refers to ambient temperature, which may be 20°C-30°C; in some embodiments, 22°C-28°C; in some embodiments, 24°C-26°C; in some embodiments, 25°C.

[0105] In the above text of the present invention, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. Based on the disclosed numbers, the value of each number may vary by less than ±10% or reasonable differences considered by those skilled in the art, such as ±1%, ±2%, ±3%, ±4% or ±5%.

[0106] The term "optional", "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. For example, "optional surfactant" means that a surfactant may or may not be present.

[0107] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0108] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction. DETAILED DESCRIPTION

[0109] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0110] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0111] The experimental methods without specific conditions in the examples are generally based on well-known methods in the art.

[0112] Unless otherwise specified, the "%" in each solution of the present invention indicates the mass percentage when indicating the content of the solid after dissolving the solid solute in the solvent, and indicates the volume percentage when indicating the content of the liquid solute after dissolving the liquid solute in the solvent.

[0113] Main materials and their sources

[0114]

[0115]

[0116] The amino acid sequence of the wild-type IDH1 of the present invention is as described in Seq ID NO.48, and the amino acid sequence of the wild-type IDH2 is as described in Seq ID NO.49.

[0117] Example 1: Screening of shRNA and sgRNA target sequences that inhibit D2HGDH expression

[0118] 1. Construction of recombinant plasmid

[0119] 1.1 Construction of shRNA recombinant plasmid

[0120] 1.1.1 shRNA primer design

[0121] There are many websites for designing shRNA targeting sequences, which mainly use the BLAST function in the NCBI website for sequence alignment and score the interference effect of the targeting sequence. In the present invention, siDESIGN Center (https: / / horizondiscovery.com / en / ordering-and-calculation-tools / sidesign-center) is used to design the targeting sequence. The primer of shRNA consists of the selected targeting sequence, linker and neck loop structure, and the final primer sequence is shown in Table 1.

[0122] Table 1: shRNA primer sequences and their target sequences

[0123]

[0124]

[0125] 1.1.2 Primer annealing

[0126] Before connecting the primer to the vector, the primer needs to be annealed to make the single-stranded DNA primer form a double-stranded DNA fragment. The annealing system is shown in the following table:

[0127]

[0128] 1.1.3 Preparation of vector

[0129] The pLentiLox3.7 vector was double-digested with enzymes (XhoⅠ and HpaⅠ). The enzyme digestion reaction system is shown in the following table:

[0130]

[0131] After the enzyme digestion is completed, the digested vector is separated by agarose gel electrophoresis and recovered and purified using the commercial product glassmilk.

[0132] 1.1.4 Annealing primer and vector connection and transformation

[0133] In the present invention, T4 ligase is used to connect the vector and the annealing product, and the connection reaction system is as follows:

[0134]

[0135] After the T4 ligase is used to connect, add 200 μL of competent cells to the ligation product, place on ice for 20 minutes, heat shock in a 42°C water bath for 45 seconds, place the competent cells after the heat shock on ice for 2 minutes, and then evenly spread them on the solid LB medium. Place the plate upside down in a 37°C bacterial incubator and culture for about 12 hours. When clearly visible colonies grow on the plate, they can be selected for identification.

[0136] 1.1.5 Screening of positive clones

[0137] When screening correctly connected recombinant plasmid monoclonal colonies, colony PCR can be used for detection. This detection method mainly uses specific primers of the target fragment or specific primers on the vector, and uses the picked monoclonal colonies as templates for PCR amplification. Positive clones can amplify DNA fragments with the same size as the set fragments, and candidate positive clones can be identified by agarose gel electrophoresis. Specifically, the colonies on the LB plate are picked out with a 0.5-10μL pipette tip and transferred to 50μL of liquid LB with corresponding resistance for short-term expansion (37℃, 4h), and PCR amplification is performed using the bacterial solution as a template.

[0138] The colony PCR reaction system is shown in the following table:

[0139]

[0140] The PCR reaction program is shown in the following table:

[0141]

[0142] After the PCR reaction is completed, agarose gel electrophoresis is performed to determine the possible correct clones based on the molecular weight of the PCR product. 2-3 candidate positive clones are selected and the bacterial solution after short-term expansion culture is expanded again (expanded in 50mL liquid LB for 12h) to extract enough plasmid DNA for later experiments.

[0143] 1.1.6 Plasmid extraction

[0144] Plasmids in Escherichia coli can be extracted and purified using alkaline lysis. The principle is: alkaline solution can lyse Escherichia coli, and the nucleic acid substances (genomic DNA and plasmid DNA) released after lysis are in a denatured state. At this time, add an acidic solution to adjust the solution to neutral, and the nucleic acid substances will renature. Since genomic DNA is linear DNA and its length is much longer than plasmid DNA, it cannot maintain a normal folding state during the rapid denaturation and renaturation process, resulting in renaturation failure and precipitation. Compared with genomic DNA, plasmid DNA has a small molecular weight and is circular. It can still return to its original soluble state during rapid denaturation and renaturation, so it can be dissolved in the solution. Based on this difference between genomic and plasmid DNA, plasmid DNA can be separated and purified. The specific steps are as follows:

[0145] ① Transfer the cultured bacterial suspension to a 50 mL centrifuge tube, centrifuge at 10,000 g for 5 min, and discard the supernatant;

[0146] ② Add 2.0 mL of pH 8.0 P1 aqueous solution (containing a final concentration of 50 mM Tris-HCl (pH 8.0), a final concentration of 10 mM EDTA, and a final concentration of 100 μg / mL RNase) and resuspend until there are no lumps;

[0147] ③ Add 2.0 mL of P2 aqueous solution (containing a final concentration of 0.2 M NaOH and a final concentration of 10% SDS) and gently shake to allow the bacteria to fully lyse in an alkaline environment, release nucleic acid substances and denature them;

[0148] ④ Add 2.0 mL of P3 aqueous solution (containing KAc with a final concentration of 3 M and glacial acetic acid with a final concentration of 11.5%) and mix well to neutralize the alkaline substances in the P2 aqueous solution, restore the solution to a neutral environment, and allow the nucleic acid substances to renature;

[0149] ⑤ After standing for 10 minutes, centrifuge at 10,000 g for 10 minutes, and transfer the supernatant to a new centrifuge tube; add 6.0 mL of isopropanol to the supernatant, mix thoroughly to precipitate the plasmid, stand for 10 minutes to allow the plasmid to fully precipitate, centrifuge at 10,000 g for 10 minutes, remove the supernatant, and turn upside down to dry;

[0150] ⑥ Add 500 μL TE aqueous solution (containing a final concentration of (10 mM Tris-HCl pH 8.0) and a final concentration of 1 mM EDTA) to dissolve the precipitated plasmid. After the plasmid precipitate is completely dissolved, add 1.5 mL of guanidine hydrochloride and let it stand for 10 minutes to remove protein impurities in the solution;

[0151] ⑦ Use DNA adsorption column to purify the plasmid. Before use, add 200μL TE aqueous solution to the DNA adsorption column for 5min-10min to moisten the column, and centrifuge at 10000g for 2min;

[0152] ⑧ Pass the liquid in the centrifuge tube through the column three times, 700 μL each time, centrifuge at 10000 g for 1 min each time;

[0153] ⑨After all plasmid DNA is adsorbed, use New Wash (containing EDTA with a final concentration of 1mM, NaCl with a final concentration of 0.1M, Tris-HCl with a final concentration of 10mM (pH7.5) and anhydrous ethanol with a final concentration of 50% (v / v), and the balance is water) to remove non-specific binding impurities. Add 600μL each time, 10000g, centrifuge for 1min, and centrifuge again after washing to remove residual ethanol;

[0154] ⑩ Take out the inner tube of the adsorption column and put it in a new 1.5mL centrifuge tube. Take 80μL TE aqueous solution (containing Tris-HCl (pH 8.0) with a final concentration of 10mM and EDTA with a final concentration of 1mM) and carefully add it so that the liquid covers the adsorption membrane at the bottom of the adsorption column. Let it stand for at least 30 minutes to fully dissolve the plasmid. Centrifuge at 10000g for 1 minute to collect the purified plasmid DNA. Measure the concentration and set aside.

[0155] To further confirm whether the connected recombinant plasmid is correct, 10 μg of plasmid was sent to a sequencing company for DNA sequencing to ensure that the connected DNA fragment did not produce mutations.

[0156] 1.2 Construction of sgRNA recombinant plasmid

[0157] When constructing the sgRNA recombinant plasmid, except that the vector was replaced with lentiCRISPR v2, the restriction enzyme was replaced with Bsm BI, and the primer sequence was replaced with the primers shown in Table 2, the other conditions were the same as those for constructing the shRNA recombinant plasmid.

[0158] Table 2: sgRNA primer sequences and their target sequences

[0159]

[0160]

[0161] 2. Lentivirus packaging and virus infection of U251 cell line

[0162] 2.1 Lentivirus packaging

[0163] Taking HEK-293T cells cultured in 6-well plates for virus packaging as an example, the lentivirus packaging steps are as follows:

[0164] ① Divide HEK-293T cells into 6-well plates for culture. The cell density required for virus packaging is about 90%. Before formally packaging the lentivirus, the medium needs to be changed 2-3 hours in advance, so that the cells can be kept in a better state, which is conducive to virus packaging;

[0165] ② Use PEI transfection reagent with a molecular weight of 40000 to transfect the plasmid required for virus packaging (the recombinant plasmid constructed in "1. Construction of recombinant plasmid" in Example 1) and the lentiviral packaging auxiliary plasmid (PMDL:VSVG:REV) into HEK-293T cells. The transfection system is: 180μL HBS buffer (containing a final concentration of 100mM NaCl, a final concentration of 10mM HEPES (pH 7.4), and the balance is water), 1.5μg core plasmid (shRNA recombinant plasmid or sgRNA recombinant plasmid), 1.5μg Mix (PMDL:VSVG:REV=5:3:2 (mass ratio)) and 20μL 10μM PEI (HBS dissolved) solution, mix well and let stand for 20min;

[0166] ③ Slowly and evenly add the above transfection mixture to the culture dish, mix well, and place in the incubator for culture. After 4-8 hours, replace the cells with 2mL of fresh culture medium;

[0167] ④ After culturing for 36 hours, if the cells are still in good condition, add 1 mL of fresh culture medium and continue culturing for 24 hours.

[0168] ⑤ After about 60 hours, there are a large number of "pseudovirus" particles secreted in the culture medium. Collect the culture medium into a 2 mL freezing tube, centrifuge at 3000 rpm for 5 minutes, and sink the HEK-293T cells suspended in the culture medium to the bottom of the tube. Transfer the supernatant, aliquot the virus, and store it at -80°C for later use.

[0169] 2.2 Lentivirus infection of U251 cell line

[0170] Mix the virus obtained in step ⑤ of "2.1 Packaging of Lentivirus" with the cell culture medium containing U251 cell line for infection. When infecting cells, the cell density needs to be adjusted according to the growth rate of cells. Generally, it is appropriate to allow cells to grow fully 24 hours after infection with the virus. Replace the cells with fresh culture medium before infection with the virus. To improve the efficiency of virus infection, the negative charge removal reagent polybrene (final concentration of 10μg / mL) can be added to remove the negative charge on the surface of cells and virus particles to reduce electrostatic repulsion. The culture medium can be replaced or passaged 24 hours after infection with the virus.

[0171] 3. siRNA transfection of U251 cell line

[0172] siRNA can be transfected into cells through liposome transfection reagent to perform the function of knocking down the expression of the target gene. 1 μg of the synthesized siRNA shown in Table 3, 3 μL of Lipo liposome 3000 and 200 μL of serum-free DMEM were mixed and allowed to stand for 20 minutes. Then, they were added to U251 cells cultured in a 6-well plate at 50% density.

[0173] Table 3: siRNA sequences

[0174] name sequence siRFP Seq ID NO.33: 5′-GAAAUCCACCCGGGGCGCCC-3′ siRNA-1 (siD2HGDH-1) Seq ID NO.10: 5′-GGAAGGACAACACGGGCUA-3′ siRNA-2 (siD2HGDH-2) Seq ID NO.11: 5′-GGGGAAACGUGGCAACCAA-3′ siRNA-3 (siD2HGDH-3) Seq ID NO.12: 5′-GGGCUGUGAACGUGGCUUU-3′ siRNA-4 (siD2HGDH-4) Seq ID NO.34: 5′-UUUACGUCCUCAUCGAGACUU-3′ siRNA-5 (siD2HGDH-5) Seq ID NO.35: 5′-UCGGGUUCUGCUCCUACAAAG-3′ siRNA-6 (siD2HGDH-6) Seq ID NO.36: 5′-GGCCUGCGGUUUCUUCGAUAU-3′

[0175] 4. Western blot detection of D2HGDH protein expression

[0176] Western blot (WB) is a protein quantitative detection method that combines polyacrylamide gel electrophoresis, protein transfer technology, antigen-antibody adsorption technology, enzyme-linked antibody technology, and exposure and development technology. Its main steps are as follows:

[0177] ①Prepare protein samples. Protein samples that need to be tested by WB mainly include cultured cells and animal tissues. When preparing samples, for cultured cells, aspirate the culture medium and add an appropriate amount of cell lysis solution containing protease inhibitors, then scrape the cells from the culture dish, transfer them to a new 1.5mL centrifuge tube, ultrasonically disrupt, and centrifuge at 4℃12000g for 10min. For animal tissue samples, homogenize after adding cell lysis solution, ultrasonically disrupt, and centrifuge at 4℃12000g for 10min. Transfer the supernatant to a new 1.5mL centrifuge tube. Take 2μL and use G250 Coomassie Brilliant Blue staining to detect the total protein concentration. According to the detected protein concentration, normalize the samples to make the total protein concentration of each sample consistent (3-5μg / μL). Add 5×SDS sample preparation buffer and heat at 100℃ for 10min to obtain the WB protein sample.

[0178] ② Denaturing gel polyacrylamide gel electrophoresis. Prepare polyacrylamide gel of different concentrations (8-15%) according to the molecular weight of the target protein. After preparation, set it up with an electrophoresis rack, put it in a vertical electrophoresis tank, and add electrophoresis buffer. According to the experimental design, add the protein samples to the lanes of the gel in sequence. Separate the protein samples at a voltage of 100V, and determine the running time according to the size of the target protein under the instructions of the pre-made protein maker (usually 60-120min);

[0179] ③ Protein transfer. According to the size of the target protein, cut out a PVDF membrane of appropriate size. Before transfer, the PVDF membrane needs to be activated with ethanol. After placing the negative electrode, filter paper, gel, PVDF membrane, filter paper, and anode in order, put the transfer clip into the vertical electrophoresis tank, add transfer buffer, and transfer at 100V for 30-90min. To avoid the buffer temperature being too high during the transfer process, add a pre-cooled ice pack to the electrophoresis tank. If the transfer time exceeds 60min, the ice pack needs to be replaced.

[0180] ④ Incubate with antibodies. Before incubating with antibodies, block the area on the PVDF membrane that has not been transferred to the protein with 5% skim milk or 5% BSA. After washing with TBS-T three times, incubate with primary antibodies (anti-D2HGDH, anti-IDH1 R132H, anti-Flag or anti-GAPDH) and secondary antibodies (GoatAnti-Rabbit IgG Antibody or GoatAnti-Mouse IgGAntibody) in sequence.

[0181] ⑤ Exposure and development. After washing the secondary antibody with TBS-T, add the exposure solution mixed with ECLA and ECLB to the PVDF membrane, quickly put the PVDF membrane soaked in the exposure solution into the exposure clip covered with plastic wrap, use film to collect the signal in the darkroom, and then develop and fix the film. After washing the film, dry it.

[0182] Main reagent formula:

[0183] 10% separation gel: 66.8 mL 30% Acr-Bis, 15.8 mL 65% sucrose, 20 mL 10× lower buffer (containing a final concentration of 3.5 M Tris-HCl (pH 8.8) and 1% SDS), dilute to 200 mL with water;

[0184] 4% stacking gel 26.6 mL 30% Acr-Bis, 50 mL 4× stacking buffer (containing Tris-HCl (pH 6.8) with a final concentration of 0.5 M and 0.4% SDS), dilute to 200 mL with water.

[0185] Electrophoresis buffer: 25 mM Tris base, 0.2 M glycine, 1.0% SDS;

[0186] Transfer buffer: 25 mM Trisbase, 0.2 M glycine, 10% anhydrous ethanol;

[0187] TBS-T: 0.1% Tween-20, 0.242% Tris base, 0.8% NaCl pH 7.6;

[0188] ECLA: 0.396mM P-coumaric, 25μM Luminol, 100mM Tris-HCl (pH 8.5);

[0189] ECLB: 5.4mM H2O2, 100mM Tris-HCl (pH 8.5).

[0190] 5. Screening of effective sequences for knocking down or knocking out D2HGDH

[0191] Western blot analysis was performed to analyze the expression of D2HGDH in U251 cells infected with viruses expressing shRNA or sgRNA or transfected with siRNA 72 h later. The shRNA, sgRNA or siRNA sequences with the best knockdown or knockout effect of D2HGDH were screened based on the Western blot results.

[0192] Results: The final results of Example 1 are as follows Figure 1 shown. Figure 1 In A, shRFP is a negative control, and its sequence targets the red fluorescent protein (RFP) gene of Branchiostoma floridae. Figure 1 A The results showed that shRNA-1, shRNA-2 and shRNA-3 had good knockdown effects, while shRNA-4, shRNA-5 and shRNA-6 had no significant knockdown effects.

[0193] Figure 1 The siRFP in B is a negative control, and its sequence is an RNA sequence targeting the red fluorescent protein (RFP) gene of Branchiostoma floridae. Figure 1 B The results showed that siRNA-1, siRNA-2 and siRNA-3 had good knockdown effects, while siRNA-4, siRNA-5 and siRNA-6 had no significant knockdown effects.

[0194] Figure 1 The sgCTRL in C is a negative control, which is a Lenti V2 plasmid vector that does not contain the sgRNA sequence. Figure 1 C The results showed that sgRNA-1, sgRNA-2 and sgRNA-3 had good knockout effects, while sgRNA-4, sgRNA-5 and sgRNA-6 had no significant knockout effects.

[0195] Example 2: Effect of knocking down D2HGDH expression on proliferation of IDH1 or IDH2 wild-type and mutant cells

[0196] The present invention uses an IDH1 wild-type glioma cell line U251 (abbreviated as U251 IDH1 WT), an IDH1 wild-type glioma cell line U118 (abbreviated as U118 IDH1 WT), an IDH1 R132H mutant glioma cell line U251 (abbreviated as U251 IDH1R132H), an IDH1 R132H mutant glioma cell line U118 (abbreviated as U118 IDH1R132H), an IDH2 wild-type leukemia cell line JURKAT (abbreviated as JURKAT IDH2 WT), an IDH2 R140Q mutant leukemia cell line JURKAT (abbreviated as JURKAT IDH2 R140Q), and primary IDH mutation chondrosarcoma cells HT1080 (carrying IDH1 R132C mutation) and SW1353 (carrying IDH2 R172S mutation) and other cells were used as experimental subjects for research.

[0197] 2.1 Construction of stable cell lines overexpressing wild-type IDH1 or IDH2 (IDH1 WT or IDH2 WT) and overexpressing mutant IDH1 or IDH2 (IDH1R132H or IDH2 R140Q)

[0198] Primers for specific amplification of IDH1 or IDH2 genes were designed (primer sequences were: forward primer for amplification of IDH1: Seq ID NO.52: 5′-AGAGAATTCGGATCCATGTCCAAAAAAATCAGT-3′, reverse primer: Seq ID NO.53: 5′-CTTCCATGGCTCGAGAAGTTTGGCCTGAGCTAG-3′. Forward primer for amplification of IDH2: Seq ID NO.54:

[0199] 5′-AGAGAATTCGGATCCATGGCCGGCTACCTGCGG-3′, reverse primer: Seq ID NO.55: 5′-CTTCCATGGCTCGAGCTGCCTGCCCAGGGCTCT-3′), and the cDNA obtained from U251 cells as experimental materials was used as a template to amplify the gene fragment of IDH1 or IDH2. Then, the plasmid plv-EF1-puro-Flag-IDH1 WT overexpressing IDH1 WT or the plasmid plv-EF1-puro-Flag-IDH2 WT overexpressing IDH2 WT was constructed using the same method as in Part 1 of Example 1.

[0200] On this basis, the overexpression plasmid plv-EF1-puro-Flag-IDH1 R132H carrying the IDH1 R132H mutation or the overexpression plasmid plv-EF1-puro-Flag-IDH2 R140Q carrying the IDH2 R140Q mutation was obtained by using the primer-designed point mutation cloning technology. The forward primer for IDH1 R132H was constructed: Seq ID NO.56: 5′-ATAGGTCATCATGCTTATGGGGATC-3′, and the reverse primer was constructed: Seq ID NO.57: 5′-AGCATGATGACCTATGATGATAGGT-3′. The forward primer for IDH2 R140Q was constructed: Seq ID NO.58: 5′-CTATCCGGAACATCCTGGGGGGG-3′, and the reverse primer was constructed: Seq ID NO.59: 5′-TGTTCCGGATAGTTCCATTGGGAC-3′;

[0201] Six cell lines, U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, and JURKAT IDH2 R140Q, were constructed by lentiviral infection using the same method as in Part 2 of Example 1. Using Western blot, we detected the expression of wild-type and mutant IDH1 or IDH2 (Flag), as well as the expression of D2HGDH in the above six cell lines.

[0202] To further confirm that the constructed IDH1 mutant or IDH2 mutant cell lines have the basic characteristics of IDH1 mutant or IDH2 mutant cells, we used the D-2HG detection kit to analyze the level of D-2HG in the culture medium of the above 6 cells. The specific steps are as follows:

[0203] ① Digest the above 6 cells with trypsin and count the cells. Take 1.0×10 5 cells, and plated them into 12-well plates, with 3 biological parallels set for each cell;

[0204] ②After 24 hours of culture, replace 1 mL of cell culture medium;

[0205] ③ After continuing to culture for 72 hours, take 100uL of cell culture medium into a 200μL centrifuge tube, use the incubation function of the PCR instrument, incubate at 100℃ for 20min, and after the incubation, centrifuge at 1000rpm for 10min, transfer the supernatant for later use;

[0206] ④Take 10uL of supernatant and perform the test according to the instructions of the D-2HG test kit;

[0207] ⑤ The protein concentration of the cultured cells was detected according to the method in Part 4 of Example 1 for normalization of the D-2HG concentration, and Western blot samples were prepared for analysis of the expression of IDH1 WT protein, IDH1 R132H protein, IDH2WT protein, IDH2 R140Q protein and D2HGDH protein.

[0208] Results: The experimental results are as follows Figure 2 shown.

[0209] Conclusion: The results show that IDH1 WT protein, IDH1 R132H protein, IDH2 WT protein, and IDH2 R140Q protein are expressed in U251, U118, and JURKAT cells, and the expression levels of wild type and mutant are comparable. At the same time, the protein expression level of D2HGDH is significantly increased in cells with IDH1 mutation or IDH2 mutation. Cells with IDH1 mutation or IDH2 mutation may be more dependent on the function of D2HGDH. This experimental result is one of the important theoretical foundations of the present invention. In addition, cells overexpressing IDH1 R132H or IDH2 R140Q can produce high concentrations of D-2HG. This shows that the constructed IDH1 mutant or IDH2 mutant cell lines have the basic characteristics of IDH1 mutant or IDH2 mutant cells and can be used for research related to IDH mutations.

[0210] 2.2 Cell proliferation curve analysis of knockdown of D2HGDH expression on the proliferation of IDH1 or IDH2 wild-type and mutant cells

[0211] The present invention is illustrated by taking 3 pairs of shRNAs that can effectively inhibit D2HGDH as an example. As described in Example 1, after obtaining the best shRNA sequences (shRNA-1, shRNA-2 and shRNA-3), U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HT1080 and SW1353 cell lines that knock down D2HGDH are constructed by lentiviral infection. In the present invention, a cell counter is used to count cells to draw a cell proliferation curve. The steps are as follows:

[0212] ① Digest the cells to be tested for proliferation with trypsin and then count the cells. Take 10,000 cells and spread them into a 12-well plate, setting up 3 biological parallels for each cell;

[0213] ② Count the cells every 24 hours for a total of 120 hours;

[0214] Results: The experimental results of this embodiment are as follows Figure 3 shown.

[0215] Conclusion: The results of this experiment show that knocking down the expression of D2HGDH in IDH1 mutant or IDH2 mutant cell lines can significantly inhibit the proliferation of cells. However, knocking down the expression of D2HGDH in IDH1 wild-type or IDH2 wild-type cells has limited effect on their cell proliferation ability. This indicates that knocking down D2HGDH can specifically inhibit the proliferation ability of IDH1 mutant or IDH2 mutant cells.

[0216] Example 3: Effect of D2HGDH enzyme activity on proliferation of IDH1 or IDH2 wild-type and mutant cells

[0217] In view of the fact that the main function of D2HGDH is to catalyze D-2HG, the present invention further describes the effect of D2HGDH enzyme activity on the cell proliferation of IDH1 wild-type cells, IDH2 wild-type cells, IDH1 mutant cells, and IDH2 mutant cells.

[0218] 3.1 Construction of wild-type and enzyme activity-deficient D2HGDH overexpression plasmids

[0219] By designing primers for specific amplification of the D2HGDH gene (forward primer for amplifying D2HGDH: Seq ID NO.60:

[0220] 5′-AGAGAATTCGGATCCATGCTGCCCCGTCGGCCT-3′, reverse primer: Seq ID NO.61: 5′-CTTCCATGGCTCGAGGGCCTGGCTGGGCAGC-3′), and the cDNA obtained from U251 cells as the experimental material was used as a template to amplify the gene fragment of D2HGDH. Then, the plasmid plv-EF1-puro-Flag-D2HGDH WT overexpressing wild-type D2HGDH was constructed using the same method as in Part 1 of Example 1.

[0221] To study the function of D2HGDH enzyme activity in IDH1 mutation or IDH2 mutation cells, we need to construct a plasmid with D2HGDH enzyme activity deficiency. Clinically, D-dihydroxyglutaric aciduria type II is a genetic disease caused by the loss of catalytic activity of the D2HGDH gene mutation, resulting in abnormal increase of D-2HG in the body. According to existing literature reports, we selected the D2HGDH G477R mutant as a mutant with D2HGDH enzyme activity deficiency. Using the point mutation cloning technology based on primer design, we constructed the plv-EF1-puro-Flag-D2HGDH G477R mutant plasmid. The forward primer for constructing D2HGDH G477R: Seq ID NO.62: 5′-GCGCGGAGCACCGAGTGGGC-3′, the reverse primer: Seq ID NO.63: 5′-CTGAAGCCCACTCGGTGCTC-3′.

[0222] 3.2 Cell proliferation curve analysis of the effect of D2HGDH enzyme activity on the proliferation of IDH1 or IDH2 wild-type and mutant cells

[0223] In order to study the effect of D2HGDH enzyme activity on the proliferation of wild-type and mutant IDH1 or IDH2 cells, we constructed a knockdown D2HGDH by lentiviral infection (using shRNA-1 lentivirus to knock down D2HGDH) and then complemented the wild-type D2HGDH (rWT) U251 IDH1 WT, U118 IDH1 WT, U118 IDH1 WT, JURKAT IDH2 WT, HT1080 and SW1353 cell lines, and D2HGDH enzyme activity-deficient D2HGDH mutant (rG477R) U251 IDH1 R132H, U118 IDH1 R132H, JURKAT IDH2 R140Q, HT1080 and SW1353 cell lines. Use a cell counter to count cells to draw a cell proliferation curve. The steps are as follows:

[0224] ① Digest the cells to be tested for proliferation with trypsin and then count the cells. Take 10,000 cells and spread them into a 12-well plate, setting up 3 biological parallels for each cell;

[0225] ② Count the cells every 24 hours for a total of 120 hours.

[0226] Results: The experimental results of this embodiment are as follows Figure 4 shown.

[0227] Conclusion: The results of this experiment show that the decrease in cell proliferation ability caused by knocking down the expression of D2HGDH in IDH1 mutant or IDH2 mutant cell lines can be restored to a level equivalent to the control group shRFP by complementing the wild-type D2HGDH. However, complementing the D2HGDH with missing enzyme activity cannot be restored to a level equivalent to the control group shRFP. This shows that the decrease in the proliferation ability of IDH1 mutant or IDH2 mutant cells caused by knocking down D2HGDH is mainly caused by the loss of D2HGDH enzyme activity. Therefore, the proliferation of IDH1 mutant or IDH2 mutant cells can also be inhibited by inhibiting the enzyme activity of D2HGDH through small molecule inhibitors.

[0228] Example 4: Effect of knocking down D2HGDH expression on the tumorigenicity of IDH1 mutant cells in vivo

[0229] The HT1080 cell line is a chondrosarcoma cell line with a primary IDH1 R132C mutation, and is an important cell line resource for studying IDH1 mutation or IDH2 mutation tumors. Therefore, the present invention uses HT1080 as the research object, and uses the D2HGDH knockdown constructed in Example 3 and the HT1080 cell line complemented with wild-type D2HGDH and enzyme activity-deficient mutants to analyze the effects of D2HGDH knockdown and D2HGDH enzyme activity on the in vivo tumorigenicity of IDH1 mutant tumor cells.

[0230] 4.1 Nude mouse subcutaneous tumorigenesis assay to analyze the effects of knocking down D2HGDH expression and D2HGDH enzyme activity on the tumorigenicity of IDH1 mutant cells in vivo

[0231] Ten 6-week-old female BALB / C nude mice were randomly divided into 2 groups, 5 mice in each group. Subcutaneous tumor mouse model was established according to the following steps:

[0232] ① The constructed D2HGDH knockdown and complemented wild-type D2HGDH and enzyme activity-deficient mutant HT1080 cell lines were expanded and cultured in two 100 mm cell culture dishes.

[0233] ② Take HT1080 cells in the logarithmic growth phase, digest them with trypsin, count the cells using a cell counter, and dilute the cells to 1.0×10 7 The cell suspension of 100 cells / mL was placed on ice for later use.

[0234] ③ 2.0×10 6 Each cell (200 μL) was injected into one side of the thigh of nude mice using a 1 mL medical syringe (different cells were injected into the left and right sides), and 5 nude mice were injected with each cell type.

[0235] ④ 4-6 weeks after injection, when the largest subcutaneous tumor grows to about 1 cm, the mouse is euthanized. Take photos to record the size of the tumor. After peeling off the subcutaneous tumor, take photos to record the size of the tumor, weigh it, and measure its volume.

[0236] Results: The experimental results of this embodiment are as follows Figure 5 shown.

[0237] Conclusion: Knockdown of D2HGDH expression can significantly inhibit the subcutaneous tumorigenesis of HT1080 cells, complementation of wild-type D2HGDH can restore the subcutaneous tumorigenesis of HT1080 cells, while complementation of mutants with enzyme activity loss cannot restore the subcutaneous tumorigenesis of HT1080 cells. This indicates that D2HGDH and its enzyme activity are essential for the in vivo tumorigenesis of IDH1 mutant tumor cells. Knockdown of D2HGDH or inhibition of D2HGDH enzyme activity using small molecule inhibitors can significantly inhibit the tumorigenesis of IDH1 mutant tumor cells.

[0238] Example 5: Effects of D2HGDH inhibitors on proliferation of IDH1 or IDH2 wild-type and mutant cells

[0239] Examples 2 to 4 show that the enzymatic activity of D2HGDH plays an important role in the proliferation of tumors with IDH1 mutation or IDH2 mutation. Targeting D2HGDH may be a therapeutic target for tumors with IDH1 mutation or IDH2 mutation. In view of this, the present invention designs a scheme for high-throughput screening of D2HGDH inhibitors and screens multiple effective inhibitors from more than 8,000 small molecules.

[0240] 5.1 Construction of D2HGDH inhibitor screening system

[0241] The main function of D2HGDH is to catalyze D-2HG to produce α-KG and FADH2. The rate of this reaction can be detected by detecting the rate of FADH2 generation: FADH2 can transfer hydrogen ions to the hydrogen acceptor INT (iodonitrotetrazolium violet), and INT is reduced to generate Furazan. Furazan has a characteristic absorption peak at OD530. By detecting the change of its absorption peak, the enzyme activity of D2HGDH can be quantitatively analyzed. In order to screen drugs more effectively, we optimized the enzyme activity reaction system by adjusting the substrate concentration, pH buffer system, hydrogen acceptor INT content and reaction time in the reaction system, so that the signal-to-noise ratio of the reaction system is greater than 5, and the linear reaction time is as long as possible.

[0242] Results: The results of this example are as follows Figure 6 shown.

[0243] Conclusion: The optimal system for screening D2HGDH inhibitors is: 1.0 μg D2HGDH protein, 200 μM D-2HG, 50 μΜ FAD+, 1.5 mM INT, 0.02% castor oil (Cermophor) and 100 mM Hepes pH 8.0.

[0244] 5.2 High-throughput screening of D2HGDH inhibitors

[0245] After building a high-throughput drug screening system, the Plate&Worker automated screening system was used to screen D2HGDH small molecule inhibitors. The enzyme activity reaction system was divided into two parts: D2HGDH protein and FAD + Add the buffer system Hepes to the 384-well plate first, then add the small molecule compound to the 384-well plate (add DMSO to the first two columns and the last two columns as a negative control), so that the final concentration of the small molecule is 20μM, and place it in a 37℃ constant temperature box for 10 minutes to allow the small molecule compound to bind to the D2HGDH protein as much as possible. Finally, add the other components in the reaction system to the 384-well plate, mix and immediately detect OD530 as the initial value of the reaction OD5300. Place the 384-well plate in a 37℃ constant temperature box, and detect OD530 again after 60 minutes of reaction, and count to obtain △OD530=OD530 60 -OD5300. The inhibition efficiency of small molecule compounds on D2HGDH enzyme activity was calculated based on the detected △OD530, inhibition efficiency = 1-△OD530 小分子 ÷△OD530 DMSO Small molecules with inhibition efficiencies exceeding 50% or 80% were revalidated and half-inhibition curves were plotted.

[0246] Results: The results of this example are as follows Figure 7 As shown (due to space limitations, only the preferred results are shown here).

[0247] Conclusion: From a library of more than 8,000 small molecule compounds, we screened out ginkgolic acid (C15:1) and NSC87877, which can effectively inhibit the activity of D2HGDH protein.

[0248] 5. Effects of 3D2HGDH inhibitors on proliferation of IDH1 or IDH2 wild-type and mutant cells

[0249] We treated U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2WT, JURKAT IDH2 R140Q, HuCCT1 IDH1 WT, HuCCT1 IDH1 R132H, HT1080 and SW1353 cell lines with gradient concentrations of Ginkgolic acid (C15:1) and NSC87877. The specific steps are as follows:

[0250] ① 1.0×10 4 Cells were plated into 12-well plates;

[0251] ② After the cells adhered for 24 hours, the culture medium was replaced with a series of gradient concentrations of Ginkgolic acid (C15:1) and NSC87877 for culture. The concentrations of the compounds used were: 0 (DMSO), 5 μM, 10 μM, 20 μM and 50 μM;

[0252] ③ After 24h and 120h of treatment with small molecule compounds, the cell numbers at these two time points were recorded as N1 and N2, and the cell proliferation rate = (N2-N1)÷N1.

[0253] Results: The results of this example are as follows Figure 8 and Fig. 9 shown.

[0254] Conclusion: Ginkgolic acid (C15:1) and NSC87877 can specifically inhibit the proliferation of tumor cells with IDH1 mutation or IDH2 mutation, while the inhibitory effect on IDH1 wild-type or IDH2 wild-type cells is relatively small.

[0255] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. Use of a nucleic acid molecule that specifically inhibits the transcription or translation of a D2HGDH gene in the preparation of a drug for treating a tumor, wherein the nucleic acid molecule is shRNA; The shRNA includes shRNA-1, shRNA-2 or shRNA-3; The shRNA-1 includes a sense strand and an antisense strand, the sense strand of the shRNA-1 is Seq ID NO.2, and the antisense strand of the shRNA-1 is Seq ID NO.3; The shRNA-2 includes a sense strand and an antisense strand, the sense strand of the shRNA-2 is Seq ID NO.5, and the antisense strand of the shRNA-2 is Seq ID NO.6; The shRNA-3 comprises a sense strand and an antisense strand, the sense strand of the shRNA-3 is Seq ID NO.8, and the antisense strand of the shRNA-3 is Seq ID NO.9; The target sequence of the shRNA-1 is Seq ID NO.1; The target sequence of the shRNA-2 is Seq ID NO.4; The target sequence of the shRNA-3 is Seq ID NO.7; The tumor is selected from a tumor carrying an IDH1 mutation or an IDH2 mutation; the tumor is a glioma, an acute T-cell leukemia or a chondrosarcoma.

2. Use of an inhibitor for inhibiting the activity of a protein encoded by the D2HGDH gene in the preparation of a drug for treating tumors, wherein the inhibitor for inhibiting the activity of a protein encoded by the D2HGDH gene is ginkgolic acid C15:1; the tumor is selected from a tumor carrying an IDH1 mutation or an IDH2 mutation; the tumor is a glioma, acute T-cell leukemia, chondrosarcoma or cholangiocarcinoma.

3. Use of an inhibitor for inhibiting the activity of a protein encoded by the D2HGDH gene in the preparation of a drug for treating a tumor, wherein the inhibitor for inhibiting the activity of a protein encoded by the D2HGDH gene is NSC87877; the tumor is selected from a tumor carrying an IDH1 mutation or an IDH2 mutation; the tumor is chondrosarcoma or cholangiocarcinoma.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule is shRNA; The shRNA includes shRNA-1, shRNA-2 or shRNA-3; The shRNA-1 includes a sense strand and an antisense strand, the sense strand of the shRNA-1 is Seq ID NO.2, and the antisense strand of the shRNA-1 is Seq ID NO.3; The shRNA-2 includes a sense strand and an antisense strand, the sense strand of the shRNA-2 is Seq ID NO.5, and the antisense strand of the shRNA-2 is Seq ID NO.6; The shRNA-3 comprises a sense strand and an antisense strand, the sense strand of the shRNA-3 is Seq ID NO.8, and the antisense strand of the shRNA-3 is Seq ID NO.

9.

5. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 4 and a viral vector. The recombinant vector according to claim 5 , wherein the viral vector comprises a retroviral vector, a lentiviral vector or an adenoviral vector.

7. A nucleic acid molecule delivery system, characterized in that: The nucleic acid molecule delivery system comprises the nucleic acid molecule according to claim 4.

8. The nucleic acid molecule delivery system according to claim 7, comprising at least one of N-acetylated galactosamine coupling modification, lipid nanoparticles, polymer nanoparticles and exosomes. 9 . The nucleic acid molecule delivery system according to claim 8 , wherein the polymer nanoparticles comprise polyethyleneimine or chitosan.

10. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 4 or the recombinant vector according to claim 5.

11. A composition, characterized in that It comprises at least one of the nucleic acid molecule according to claim 4, the recombinant vector according to any one of claims 5-6, the nucleic acid molecule delivery system according to any one of claims 7-9, and the host cell according to claim 10.

12. The composition according to claim 11, further comprising a pharmaceutically acceptable adjuvant.

13. The composition according to claim 12, wherein the pharmaceutically acceptable adjuvant comprises dimethyldioctadecyl ammonium and / or monophosphoric acid lipid A or aluminum adjuvant or Freund's adjuvant.

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