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CN-122005552-A - Medicine for improving expression of lysosomal related transmembrane protein 4B and screening method thereof

CN122005552ACN 122005552 ACN122005552 ACN 122005552ACN-122005552-A

Abstract

The invention relates to a medicine for improving the expression of a lysosomal related transmembrane protein 4B and a screening method thereof, belonging to the technical field of medicine screening. The invention precisely inserts fluorescent markers into the target protein LAPTM4B gene locus to realize the real-time monitoring of the dynamic change of endogenous proteins, simultaneously introduces membrane protein CD63 as an internal reference signal, utilizes a high-throughput automatic drug screening and image analysis system to quantitatively screen compounds in a large-scale drug library, finally screens out drugs capable of improving the expression of LAPTM4B, and provides a new direction for the research of the treatment targets related to the membrane-spanning protein 4B related to the subsequent lysosomes. Meanwhile, the screened medicine has good specificity, can improve the expression of LAPTM4B but does not influence CD63, so that the time-space accurate control of the LAPTM4B activity can be realized, and the method has important significance in constructing an in-vitro/in-vivo instantaneous activation model.

Inventors

  • ZHOU KECHENG
  • LIU DAN
  • LIU MINXIA

Assignees

  • 安徽医科大学

Dates

Publication Date
20260512
Application Date
20251110

Claims (10)

  1. 1. A medicament for increasing expression of a lysosomal associated transmembrane protein 4B, wherein the medicament is selected from one or more of Ruboxistaurin, motesanib, SGC0946,0946, GSK-461364, imatinib and Gefitinib.
  2. Use of ruboxistaurin, motesanib, SGC0946, GSK-461364, imatinib or Gefitinib for the preparation of a product for increasing expression of a lysosomal associated transmembrane protein 4B.
  3. 3. Use of a medicament for increasing expression of a lysosomal associated transmembrane protein 4B in the preparation of a model of cancer, wherein the medicament is selected from one or more of Ruboxistaurin, motesanib, SGC0946,0946, GSK-461364, imatinib and Gefitinib.
  4. 4. The use according to claim 3, wherein the tumour comprises hepatocellular carcinoma, breast cancer, lung cancer, gastric cancer or ovarian cancer.
  5. 5. Use of a medicament for increasing expression of a lysosomal associated transmembrane protein 4B in the preparation of a lysosomal associated transmembrane protein 4B modulator or vaccine adjuvant, wherein said medicament for increasing expression of a lysosomal associated transmembrane protein 4B is selected from one or more of Ruboxistaurin, motesanib, SGC0946, GSK-461364, imatinib and Gefitinib.
  6. 6. An agent for promoting proliferation and survival of tumor cells, wherein the agent is selected from one or more of Ruboxistaurin, motesanib, SGC0946,0946, GSK-461364, imatinib, and Gefitinib.
  7. 7. A method of screening for a drug resistant to a tumor cell, comprising administering to the tumor cell a drug that increases the expression of a lysosomal associated transmembrane protein 4B, and then adding the drug to be screened, wherein the drug that increases the expression of the lysosomal associated transmembrane protein 4B is selected from one or more of Ruboxistaurin, motesanib, SGC0946, GSK-461364, imatinib, and Gefitinib.
  8. 8. A screening method of a drug for improving the expression of a lysosomal associated transmembrane protein 4B is characterized by comprising the following steps: S1, constructing a recombinant cell for expressing a lysosomal associated transmembrane protein 4B and a lysosomal associated membrane protein 3, wherein the lysosomal associated transmembrane protein 4B is labeled with a first fluorescent protein, the lysosomal associated membrane protein 3 is labeled with a second fluorescent protein, and the first fluorescent protein is different from the second fluorescent protein; s2, incubating the medicine to be screened with the recombinant cells, detecting fluorescent signals, calculating the ratio of the fluorescent signals of the first fluorescent protein to the fluorescent signals of the second fluorescent protein in the recombinant cells, and screening to obtain the medicine for improving the expression of the lysosome related transmembrane protein 4B.
  9. 9. The method according to claim 8, wherein in step S1, the first fluorescent protein-labeled lysosomal associated transmembrane protein 4B is obtained by inserting the first fluorescent protein between the 847 th to 848 th gene sequences of the lysosomal associated transmembrane protein 4B; the second fluorescent protein marked lysosome related membrane protein 3 is obtained by inserting the second fluorescent protein between 786 th and 787 th gene sequences of the lysosome related membrane protein 3.
  10. 10. The method according to claim 8, wherein in step S1, the recombinant cells are prepared by introducing a Cas9 protein encoding gene, a sgRNA pair, and a donor DNA into a recipient cell, wherein the sgRNA pair has the sequence shown in SEQ ID NO.1-4, the donor DNA has the gene sequence shown in SEQ ID NO.5-6, and the recipient cell expresses a lysosomal associated transmembrane protein 4B and a lysosomal associated membrane protein 3.

Description

Medicine for improving expression of lysosomal related transmembrane protein 4B and screening method thereof Technical Field The invention relates to the technical field of drug screening, in particular to a drug expressed by lysosome related transmembrane protein 4B and a screening method thereof. Background Currently, global tumor drug development is rapidly moving from the traditional "cytotoxic chemotherapy" mode to the precise therapeutic stage with specific molecular targets as the core. Traditional chemotherapeutic drugs inhibit cell division primarily through nonspecific means, thereby killing tumor cells. However, such drugs often have serious toxic side effects and drug resistance problems. In recent years, along with the rapid development of genomics, structural biology and signal transduction studies, it has been increasingly recognized that tumor development and progression are a comprehensive result of abnormal activation of various driving genes (DRIVER GENES) and key signal pathways. Therefore, target-based drug screening models have gradually replaced empirical screening (EMPIRICAL SCREENING), becoming the dominant direction of modern drug discovery. The strategy can realize accurate regulation and control of tumor cells by identifying and intervening the key molecular targets, and greatly improve the curative effect and safety of the medicine. Among a number of potential molecular targets, lysosomal related transmembrane protein 4B (Lysosomal-associated transmembrane protein B, lapm 4B) has received attention in recent years as a novel oncogene. The protein was identified as a significantly high-expressing molecule in hepatocellular carcinoma (Hepatocellular carcinoma, HCC) at the earliest, and subsequent studies found that it was abnormally up-regulated in breast cancer, lung cancer, gastric cancer, ovarian cancer, and acute myeloid leukemia. LAPTM4B promotes the growth, migration, invasion and drug resistance formation of tumor cells by regulating and controlling autophagy pathway (autophagy), mTorrC 1 signal activation, drug excretion, membrane protein transport and other processes. Its high expression level is significantly associated with poor prognosis for a variety of cancer patients. However, to date, no regulatory molecule directed against lapm 4B has been reported, and the lack of an efficient, systematic screening platform has become an important bottleneck in this field. In view of the key role of LAPTM4B in tumorigenesis and drug tolerance, it is necessary to establish a small molecule screening system capable of dynamically monitoring its expression, localization and regulation at the cellular level, thereby screening regulatory molecules against LAPTM 4B. Disclosure of Invention The technical problem underlying the present invention is to overcome the lack of a regulatory molecule for LAPTM4B in the prior art. In order to solve the technical problems, the invention provides a lysosomal related transmembrane protein 4B agonist and a screening method thereof. The invention precisely inserts fluorescent markers into the target protein LAPTM4B gene locus to realize the real-time monitoring of the dynamic change of endogenous protein, simultaneously introduces membrane protein CD63 as an internal reference signal, utilizes a high-throughput automatic drug screening and image analysis system to quantitatively screen compounds in a large-scale drug library, and finally screens out drugs capable of improving LAPTM4B expression. By utilizing the screening method, the invention screens out the medicine capable of improving the related transmembrane protein 4B of the lysosome and provides a new direction for the subsequent research of the related therapeutic target of the related transmembrane protein 4B of the lysosome. Meanwhile, the screened medicine has good specificity, can improve the expression of LAPTM4B but does not influence CD63, so that the time-space accurate control of the LAPTM4B activity can be realized, and the method has important significance in constructing an in-vitro/in-vivo instantaneous activation model. It is a first object of the present invention to provide a medicament for increasing the expression of a lysosomal associated transmembrane protein 4B (lapm 4B), said medicament being selected from one or more of Ruboxistaurin, motesanib, SGC0946,0946, GSK-461364, imatinib and Gefitinib. Further, ruboxistaurin (CAS number 169939-94-0) is an orally active, selective PKC beta inhibitor. Motesanib (CAS number 453562-69-1) is a potent ATP-competitive inhibitor of VEGFR 1/2/3. SGC0946 (CAS number 1561178-17-3) is a selective DOT1L inhibitor. GSK-461364 (CAS number 929095-18-1) is a selective, reversible, ATP-competitive PLK1 inhibitor. Imatinib (CAS number 152459-95-5) is a tyrosine kinase inhibitor that selectively inhibits BCR/ABL, v-ABL, PDGFR, c-kit kinase activity. Gefitinib (CAS number 18475-35-2) is an EGFR tyrosine kinase inhibitor. A second object of the pre