CN112675169A

CN112675169A - Application of ITM-1B4 in preparation of drugs for preventing and/or treating tumors

Info

Publication number: CN112675169A
Application number: CN202011543184.8A
Authority: CN
Inventors: 夏培雪; 高凯瑜; 刘颖
Original assignee: Nanjing Jingruikang Molecular Medicine Technology Co ltd
Current assignee: Nanjing Jingruikang Molecular Medicine Technology Co ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-04-20

Abstract

The invention discloses a new application of ITM-1B 4. The new application comprises the following steps: 1) inhibits the interaction of KLHL22 with DEPDC 5; 2) preparing a product that inhibits the interaction of KLHL22 with DEPDC 5; 3) inhibiting proliferation of eukaryotic tumor cells; 4) preparing a eukaryotic tumor cell proliferation inhibitor; 5) preventing and/or treating tumors; 6) preparing a product for preventing and/or treating tumors. The established KLHL22-DEPDC5 interaction model is used for screening ITM-1B4, and when the ITM-1B4 cells are added, the KLHL22-DEP interaction strength is obviously lower than that of a control group. The anti-cancer effect of ITM-1B4 is verified by using typical triple negative breast cancer cells MDA-MB-231, and the result shows that the number of cells added into a drug group is far lower than that of a control group, which indicates that the compound has the effect of inhibiting the growth of triple negative breast cancer cells.

Description

Application of ITM-1B4 in preparation of drugs for preventing and/or treating tumors

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of ITM-1B4 in preparation of a medicine for preventing and/or treating tumors.

Background

KLHL22 is an important E3 ubiquitination ligase, and the substrates of the ligase comprise PLK1, DEPDC5, PD-1 and the like, and have important regulation and control functions in the aspects of cell growth, proliferation and cancer formation. In previous reports, KLHL22 activated mTOR signaling pathway via ubiquinated DEPDC5 to promote the proliferation of triple negative breast cancer. Therefore, it is desirable to develop a drug capable of inhibiting the KLHL22-DEPDC5 interaction to retard the growth of breast cancer cells.

Disclosure of Invention

The invention aims to provide a new application of a compound ITM-1B4 or a pharmaceutically acceptable salt thereof.

The compound ITM-1B4, SMILES structural formula:

N1＝C(NC＝2C＝CC(＝CC1＝2)N＝CC＝3C＝CC＝CC＝3)C＝4C＝CC＝CC＝4

the structural formula is shown as formula I:

the new application of the compound ITM-1B4 or the pharmaceutically acceptable salt thereof provided by the invention is the application thereof in any one of the following:

1) inhibits the interaction of KLHL22 with DEPDC 5;

2) preparing a product that inhibits the interaction of KLHL22 with DEPDC 5;

3) inhibiting proliferation of eukaryotic tumor cells;

4) preparing a eukaryotic tumor cell proliferation inhibitor;

5) preventing and/or treating tumors;

6) preparing a product for preventing and/or treating tumors.

The eukaryote is a human or a mammal; the tumor cell is a cancer cell; the cancer cell is a breast cancer cell, and specifically can be a triple negative breast cancer cell MDA-MB-231.

The tumor is a carcinoma; the cancer is specifically breast cancer, and further may be triple negative breast cancer.

The product can be a medicine.

The following products prepared by taking ITM-1B4 or pharmaceutically acceptable salts thereof as active ingredients also belong to the protection scope of the invention; the product has any one of the following uses:

a) inhibition of KLHL22 interaction with DEPDC 5;

b) inhibiting tumor cell proliferation;

c) preventing and/or treating tumors.

When necessary, one or more pharmaceutically acceptable carriers can be added into the product; the carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The above medicine can be made into various forms such as injection, tablet, powder, granule, capsule, oral liquid, paste, cream, etc.; the medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The above drugs can be introduced into body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

In order to model the KLHL22-DEPDC5 interaction, the inventors have tried the ALPHA, Nanobit, et al system. KLHL22-DEPDC5 expressed in the prokaryotic cells was found to be unable to work on the ALPHA system. It is presumed that it is difficult for prokaryotically expressed KLHL22 and DEPDC5 to interact with each other. Similarly, the inventors have utilized the NanoBit system to construct the KLHL22-DEPDC5 system, which is also not workable. Finally, the NanoBRET screening system was chosen, in which we selected the interaction of KLHL22 with the DEP domain, where depc 5 interacts with KLHL 22.

In constructing KLHL22-DEP interaction system, the inventors constructed KLHL22 and DEP to pHTC, pHTN, pNLF1-N and pNLF1-C of Promega Nanobret, thereby obtaining 8 plasmids. And the connection of different proteins at N and C ends is tested, and finally, the pHTC-KLHL22 and pNLF1-N-DEP have the best interaction effect (figure 2). In addition to the different modes of ligation of the proteins, we also tested the transfection ratios of the plasmids and found that when pHTC-KLHL22 was found to have a plasmid amount of 2ug and pNLF1-N-DEP was found to have a plasmid amount of 0.02ug, the conditions were more suitable for drug screening (FIG. 3). In addition, we also over-expressed the ha-DEP plasmid simultaneously with the over-expression of pHTC-KLHL22 and pNLF 1-N-DEP. The NanoBRET signal of KLHL22-DEP was found to be suppressed by the overexpression of ha-DEP, indicating that the system of the present invention truly reflects the interaction of KLHL 22-DEP.

The compound ITM-1B4 was screened by using the established KLHL22-DEPDC5 interaction model, and the intensity of the KLHL22-DEP interaction was significantly lower than that of the control group (ctrl) when added to the cells of the compound ITM-1B 4. Indicating that the compound has the function of inhibiting the KLHL22-DEP interaction. The anti-cancer effect of the compound ITM-1B4 is verified by typical triple negative breast cancer cells MDA-MB-231, and the result shows that the number of the cells added into a drug group is far lower than that of a control group, which indicates that the compound has the effect of inhibiting the growth of triple negative breast cancer cells.

Drawings

FIG. 1 is a schematic diagram of the interaction between the proteins detected by the NanoBRET method; the NanoLuc generates 460nm fluorescence, activates 618ligand and fluorescence above 600nm, and the intensity of 600nm/460nm is the interaction intensity of two proteins.

Fig. 2 shows the detection of different KLHL22 and DEP connections.

FIG. 3 is a graph showing the detection of the intensity of the interaction between KLHL-HT and NL-DEP at different transfection ratios; KLHL-HT: NL-DEP: 1 mug +1 mug of A, 2 mug +0.2 mug of B, 2 mug +0.02 mug of C, 2 mug +0.002 mug of D and 2 mug +0.0 mug of negative; positive 2. mu. g P53-HT + 0.2. mu.g NL-MDM 2.

Fig. 4 shows that the signal intensity of the NanoBRET system is detected by overexpression of an empty vector or ha-DEP based on overexpression of a NanoBRET-related plasmid, and the signal intensity is significantly different from the signal intensity of the NanoBRET system, wherein p is 0.0062.

FIG. 5 is the inhibition of the KLHL22-DEP protein interaction by compound ITM-1B 4.

FIG. 6 is a graph showing the inhibitory effect of compound ITM-1B4 on MD A-MB-231.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

In the following examples: human KLHL22 (uniprot: Q53GT1), DEP (DEPDC5 DEP domain)

KLHL-HT for pHTC-KLHL 22; HT-KLHL represents pHTN-KLHL 22;

KLHL-NL for pNL1-C-KLHL 22; NL-KLHL represents pNLF1-N-KLHL 22;

DEP-NL stands for pNLF 1-C-DEP; NL-DEP stands for pNLF 1-N-DEP;

DEP-HT represents pHTC-DEP; HT-DEP stands for pHTN-DEP.

The following examples employ the methods: NanoBRET (promega, https:// www.promega.com.cn/Products/Protein-Interactions/Live-Cell-Protein-Interactions/NanoBRET-PPI-Starter-Systems /)

The compound ITM-1B4, SMILES structural formula used in the examples below:

N1＝C(NC＝2C＝CC(＝CC1＝2)N＝CC＝3C＝CC＝CC＝3)C＝4C＝CC＝CC＝4

the structural formula is shown as formula I:

this compound was purchased from ChemDiv library of compounds with catalog number: 8004-0798.

Example 1 construction of KLHL22-DEPDC5 protein interaction screening System

The experimental method comprises the following steps:

firstly, a related plasmid (promega, N1811) for over-expression of KLHL22 and DEP is constructed by using a traditional plasmid construction method, and specifically, KLHL22 and DEP are constructed on pHTC, pHTN, pNLF1-N and pNLF1-C of Nanobret, so that 8 plasmids are obtained. After the plasmid was obtained, the plasmid was transfected with PEI reagent (1mg/mL, biosciences, 24765).

The protein amino acid sequence of KLHL22 is shown as a sequence 1 in a sequence table; the amino acid sequence of the DEPDC5 protein DEP structural domain is shown as a sequence 2 in a sequence table;

the nucleotide sequence of the gene for coding the KLHL22 protein is shown as a sequence 3 in a sequence table; the nucleotide sequence of the DNA molecule for coding the DEP structural domain is shown as a sequence 4 in a sequence table.

When the 8 recombinant plasmids are constructed, the adopted enzyme cutting sites are SacII and XbaI.

pCDNA3.1-HA: available from addgene, 128034.

pCDNA3.1-HA-DEP: the DNA sequence encoding the DEP domain (as shown in sequence 4 of the sequence listing) was ligated into the pCDNA3.1-HA plasmid using BamHI and XhoI using conventional methods.

First, HEK-293T cells were grown to a suitable growth state and plated at 30% cell density in 6-well plates. One day later, 0.8-1.2 x 10 pairs⁶The number of cells was plasmid transfected 1ug (plasmid related to KLHL22 overexpression) +1ug (plasmid related to DEP overexpression). One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶Per mL, and adding

NanoBRET^TM618Ligand (1000-fold dilution, promega, N1662). After 24 hours, 25uL of luciferin substrate(s) was added to the cells

Luciferase substrate) (100-fold dilution, promega, N1662), mixed well and added to wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer Envision). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. The results are shown in FIG. 2. As can be seen from FIG. 2, pHTC-KLHL22 has the strongest bioluminescent signal compared to the pNLF1-N-DEP group (KLHL-HT + NL-DEP) compared to the other 7 groups of conditions.

In addition to the different modes of linkage of the proteins, the inventors further examined the transfection ratios of the plasmids. pHTC-KLHL22 was transfected with pNFL1-N-DEP in the same manner as A at 1. mu.g + 1. mu.g, B at 2. mu.g + 0.2. mu.g, C at 2. mu.g + 0.02. mu.g, D at 2. mu.g + 0.002. mu.g, negative at 2. mu.g +0. mu.g, or positive at 2. mu.g pHTC-TP53+ 0.2. mu.g pNLF1-N-MDM 2. First, HEK-293T cells were grown to a suitable growth state and plated at 30% cell density in 6-well plates. One day later, cells were grouped as described above for plasmid transfection. One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶Per mL, and adding

Luciferase substrate) (100-fold dilution, promega, N1662), mixed well and added to wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer Envision). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. And the intensity homogenization of the group A is adjusted to be 1, and other experimental groups are correspondingly adjusted. The results are shown in FIG. 3. In four groups A-D, 2. mu.g: 0.02 μ g group (group C) and 2 μ g: the group of 0.002. mu.g (group D) had higher strength of interaction than the positive control group (positive) given by the kit.

In FIG. 4, pHTC-KLHL22 was transfected with the pNFL1-N-DEP group at 1. mu.g + 0.01. mu.g +1ug of pCDNA3.1-HA or 1. mu.g + 0.01. mu.g +1ug of pCDNA3.1-HA-DEP. One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶one/mL and 618ligand (1000-fold dilution, promega, N1662) was added. After 24 hours, 25uL of fluorescein substrate (100 fold dilution, promega, N1662) was added to the cells, mixed well and added to the wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. The intensity of the control (control) was adjusted to 1 for uniformity and the other experimental groups were adjusted accordingly.

The results in fig. 4 show that the over-expressed ha-DEP group (o.v. ha-DEP) is able to competitively inhibit the interaction of KLHL22 with DEP.

Example 2 inhibition of the KLHL22-DEP protein interaction by the Compound ITM-1B4

HEK-293T cells were cultured to the appropriate growth state and plated at 30% cell density in 6-well plates. Transfection was performed at 2. mu.g + 0.02. mu.g using pHTC-KLHL22 and pNFL1-N-DEP plasmid. One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶Per mL, and adding

NanoBRET^TM618Ligand (1000-fold dilution, promega, N1662). After 16 hours, the cells were added with compound ITM-1B4(compound) or DMSO solvent (ctrl) at a concentration of 10. mu.M. After 8 hours, 25uL of luciferin substrate(s) was added to the cells

Luciferase substrate) (100-fold dilution, promega, N1662), mixed well and added to wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer Envision). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. And the intensity homogenization of the control (ctrl) was adjusted to 1, with corresponding adjustments for the other experimental groups. The results are shown in FIG. 5. As can be seen from FIG. 5, the KLHL22-DEP interaction intensity was significantly lower than that of the control (ctrl) in the cells added to the compound group (compound). Indicating that the compound has the function of inhibiting the KLHL22-DEP interaction.

Example 3 inhibition of triple negative breast cancer cells MDA-MB-231 by Compound ITM-1B4

Typical triple negative breast cancer cells MDA-MB-231 were seeded into 96-well plates at 5000 cells per well at 100. mu.L, and after the cells were attached, 1uL of 10mM compound (compound) or DMSO solvent (ctrl) was added per well at a final concentration of 100. mu.M. After 48 hours, the number of cells was counted, and the number of cells in the drug-added group (compound) was much lower than that in the control group (ctrl), indicating that the compound had the effect of inhibiting the growth of cancer cells.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> Nanjing Jingruikang molecular medicine science and technology Co., Ltd

Application of <120> ITM-1B4 in preparation of drugs for preventing and/or treating tumors

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 634

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Glu Glu Gln Glu Phe Thr Gln Leu Cys Lys Leu Pro Ala Gln

1 5 10 15

Pro Ser His Pro His Cys Val Asn Asn Thr Tyr Arg Ser Ala Gln His

20 25 30

Ser Gln Ala Leu Leu Arg Gly Leu Leu Ala Leu Arg Asp Ser Gly Ile

35 40 45

Leu Phe Asp Val Val Leu Val Val Glu Gly Arg His Ile Glu Ala His

50 55 60

Arg Ile Leu Leu Ala Ala Ser Cys Asp Tyr Phe Arg Gly Met Phe Ala

65 70 75 80

Gly Gly Leu Lys Glu Met Glu Gln Glu Glu Val Leu Ile His Gly Val

85 90 95

Ser Tyr Asn Ala Met Cys Gln Ile Leu His Phe Ile Tyr Thr Ser Glu

100 105 110

Leu Glu Leu Ser Leu Ser Asn Val Gln Glu Thr Leu Val Ala Ala Cys

115 120 125

Gln Leu Gln Ile Pro Glu Ile Ile His Phe Cys Cys Asp Phe Leu Met

130 135 140

Ser Trp Val Asp Glu Glu Asn Ile Leu Asp Val Tyr Arg Leu Ala Glu

145 150 155 160

Leu Phe Asp Leu Ser Arg Leu Thr Glu Gln Leu Asp Thr Tyr Ile Leu

165 170 175

Lys Asn Phe Val Ala Phe Ser Arg Thr Asp Lys Tyr Arg Gln Leu Pro

180 185 190

Leu Glu Lys Val Tyr Ser Leu Leu Ser Ser Asn Arg Leu Glu Val Ser

195 200 205

Cys Glu Thr Glu Val Tyr Glu Gly Ala Leu Leu Tyr His Tyr Ser Leu

210 215 220

Glu Gln Val Gln Ala Asp Gln Ile Ser Leu His Glu Pro Pro Lys Leu

225 230 235 240

Leu Glu Thr Val Arg Phe Pro Leu Met Glu Ala Glu Val Leu Gln Arg

245 250 255

Leu His Asp Lys Leu Asp Pro Ser Pro Leu Arg Asp Thr Val Ala Ser

260 265 270

Ala Leu Met Tyr His Arg Asn Glu Ser Leu Gln Pro Ser Leu Gln Ser

275 280 285

Pro Gln Thr Glu Leu Arg Ser Asp Phe Gln Cys Val Val Gly Phe Gly

290 295 300

Gly Ile His Ser Thr Pro Ser Thr Val Leu Ser Asp Gln Ala Lys Tyr

305 310 315 320

Leu Asn Pro Leu Leu Gly Glu Trp Lys His Phe Thr Ala Ser Leu Ala

325 330 335

Pro Arg Met Ser Asn Gln Gly Ile Ala Val Leu Asn Asn Phe Val Tyr

340 345 350

Leu Ile Gly Gly Asp Asn Asn Val Gln Gly Phe Arg Ala Glu Ser Arg

355 360 365

Cys Trp Arg Tyr Asp Pro Arg His Asn Arg Trp Phe Gln Ile Gln Ser

370 375 380

Leu Gln Gln Glu His Ala Asp Leu Ser Val Cys Val Val Gly Arg Tyr

385 390 395 400

Ile Tyr Ala Val Ala Gly Arg Asp Tyr His Asn Asp Leu Asn Ala Val

405 410 415

Glu Arg Tyr Asp Pro Ala Thr Asn Ser Trp Ala Tyr Val Ala Pro Leu

420 425 430

Lys Arg Glu Val Tyr Ala His Ala Gly Ala Thr Leu Glu Gly Lys Met

435 440 445

Tyr Ile Thr Cys Gly Arg Arg Gly Glu Asp Tyr Leu Lys Glu Thr His

450 455 460

Cys Tyr Asp Pro Gly Ser Asn Thr Trp His Thr Leu Ala Asp Gly Pro

465 470 475 480

Val Arg Arg Ala Trp His Gly Met Ala Thr Leu Leu Asn Lys Leu Tyr

485 490 495

Val Ile Gly Gly Ser Asn Asn Asp Ala Gly Tyr Arg Arg Asp Val His

500 505 510

Gln Val Ala Cys Tyr Ser Cys Thr Ser Gly Gln Trp Ser Ser Val Cys

515 520 525

Pro Leu Pro Ala Gly His Gly Glu Pro Gly Ile Ala Val Leu Asp Asn

530 535 540

Arg Ile Tyr Val Leu Gly Gly Arg Ser His Asn Arg Gly Ser Arg Thr

545 550 555 560

Gly Tyr Val His Ile Tyr Asp Val Glu Lys Asp Cys Trp Glu Glu Gly

565 570 575

Pro Gln Leu Asp Asn Ser Ile Ser Gly Leu Ala Ala Cys Val Leu Thr

580 585 590

Leu Pro Arg Ser Leu Leu Leu Glu Pro Pro Arg Gly Thr Pro Asp Arg

595 600 605

Ser Gln Ala Asp Pro Asp Phe Ala Ser Glu Val Met Ser Val Ser Asp

610 615 620

Trp Glu Glu Phe Asp Asn Ser Ser Glu Asp

625 630

<210> 2

<211> 75

<212> PRT

<213> Artificial sequence

<400> 2

Ser Thr Gly Val Gln Leu Leu Ser Glu Gln Lys Gly Leu Ser Pro Tyr

1 5 10 15

Cys Phe Ile Ser Ala Glu Val Val His Trp Leu Val Asn His Val Glu

20 25 30

Gly Ile Gln Thr Gln Ala Met Ala Ile Asp Ile Met Gln Lys Met Leu

35 40 45

Glu Glu Gln Leu Ile Thr His Ala Ser Gly Glu Ala Trp Arg Thr Phe

50 55 60

Ile Tyr Gly Phe Tyr Phe Tyr Lys Ile Val Thr

65 70 75

<210> 3

<211> 1905

<212> DNA

<213> Artificial sequence

<400> 3

atggcagagg agcaggagtt cacccagctc tgcaagttgc ctgcacagcc ctcacaccca 60

cactgcgtga acaacaccta ccgcagcgca cagcactccc aggctctgct ccgagggctg 120

ctggctctcc gggacagcgg aatcctcttc gatgttgtgc tggtggtgga gggcagacac 180

atcgaggccc atcgcatcct gctggctgcg tcctgcgatt acttcagagg aatgtttgct 240

gggggattga aggagatgga acaggaagag gtcctgatcc acggtgtgtc ctacaatgct 300

atgtgccaaa tcctacattt catatacacc tccgagctgg agctcagcct gagcaatgta 360

caagagacac tggtggctgc ctgccagctt cagatcccag aaattatcca tttctgctgt 420

gatttcctca tgtcctgggt ggacgaagag aacattctcg atgtctaccg gctggcagag 480

ctgtttgact tgagccgcct gactgagcaa ctggacacct atatcctcaa aaactttgtg 540

gccttctctc ggactgacaa gtaccgccag cttccattgg agaaggtcta ctccctcctc 600

agcagcaatc gcctggaggt ctcctgcgag accgaggtat atgagggggc ccttctctac 660

cattatagcc tggagcaggt gcaggctgac cagatctcgc tgcacgagcc cccaaagctc 720

cttgagacag tgcggtttcc gctgatggaa gctgaggtcc tgcagcggct gcatgacaag 780

ctggacccca gccctttgag ggacacagtg gccagcgccc tcatgtacca ccggaacgag 840

agcctacagc ccagcctgca gagcccgcaa acggagctgc ggtcggactt ccagtgcgtt 900

gtgggcttcg ggggcattca ctccacgccg tccactgtcc tcagcgacca ggccaagtat 960

ctaaacccct tactgggaga gtggaagcac ttcactgcct ccctggcccc ccgcatgtcc 1020

aaccagggca tcgcggtgct caacaacttc gtatacttga ttggagggga caacaatgtc 1080

caaggatttc gagcagagtc ccgatgctgg aggtatgacc cacggcacaa ccgctggttc 1140

cagatccagt ccctgcagca ggagcacgcc gacctgtccg tgtgtgttgt aggcaggtac 1200

atctacgctg tggcgggccg tgactaccac aatgacctga atgctgtgga gcgctacgac 1260

cctgccacca actcctgggc atacgtggcc ccactcaaga gggaggtgta tgcccacgca 1320

ggcgcgacgc tggaggggaa gatgtatatc acctgcggcc gcagagggga ggattacctg 1380

aaagagacac actgctacga tccaggcagc aacacttggc acacactggc tgatgggcct 1440

gtgcggcgcg cctggcacgg catggcaacc ctcctcaaca agctgtatgt gatcgggggc 1500

agcaacaacg atgccggata caggagggac gtgcaccagg tggcctgcta cagctgcacg 1560

tctggacagt ggtcatctgt ctgcccactc cctgctgggc acggtgagcc tggcattgct 1620

gtgctggaca acaggatcta tgtgttaggt ggccgctcac acaaccgcgg cagccgcaca 1680

ggctacgtgc acatttacga tgtggagaag gactgctggg aggaagggcc ccagctggac 1740

aactccatct caggcctggc ggcctgtgtg ctcaccctgc cccgctccct gctccttgag 1800

ccgccccgcg ggacccctga ccgcagccag gccgacccgg actttgcctc tgaggtgatg 1860

agtgtgtctg actgggagga gtttgacaac tccagtgagg actag 1905

<210> 4

<211> 225

<212> DNA

<213> Artificial sequence

<400> 4

tcgacaggag tccagctgct ctctgaacag aagggcctct caccgtactg cttcatcagc 60

gcggaggtgg tacactggtt ggtgaaccac gtggagggga tccagacaca ggcgatggcc 120

attgacatca tgcagaaaat gctggaagag cagctcatca cacatgcatc tggcgaagcc 180

tggcggacct tcatctacgg cttctatttc tacaagatag taacg 225

Claims

1. Use of a compound of formula I ITM-1B4 or a pharmaceutically acceptable salt thereof in any one of:

1) inhibits the interaction of KLHL22 with DEPDC 5;

2) preparing a product that inhibits the interaction of KLHL22 with DEPDC 5;

3) inhibiting proliferation of eukaryotic tumor cells;

4) preparing a eukaryotic tumor cell proliferation inhibitor;

5) preventing and/or treating tumors;

6) preparing a product for preventing and/or treating tumors.

2. Use according to claim 1, characterized in that: the eukaryote is a human or a mammal; the tumor cell is a cancer cell; the cancer cell is a breast cancer cell, and further a triple negative breast cancer cell.

3. Use according to claim 2, characterized in that: the breast cancer cell is a triple negative breast cancer cell MDA-MB-231.

4. Use according to claim 1, characterized in that: the tumor is a carcinoma; the cancer is breast cancer.

5. Use according to claim 4, characterized in that: the breast cancer is triple negative breast cancer.

6. Use according to any one of claims 1 to 5, characterized in that: the products are all medicines.

7. A product, the active ingredient of which is the compound ITM-1B4 shown in the formula I or the pharmaceutically acceptable salt thereof; the product has any one of the following uses:

a) inhibition of KLHL22 interaction with DEPDC 5;

b) inhibiting proliferation of nuclear biological tumor cells;

c) preventing and/or treating tumors.

8. The product of claim 7, wherein: the eukaryote is a human or a mammal; the tumor cell is a cancer cell; the cancer cell is a breast cancer cell, and specifically is a triple negative breast cancer cell MDA-MB-231.

9. The product of claim 7, wherein: the tumor is a carcinoma; the cancer is breast cancer, preferably triple negative breast cancer.

10. The product according to any one of claims 7-9, characterized in that: the products are all medicines.