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CN-122028940-A - PI4K inhibitor and application thereof

CN122028940ACN 122028940 ACN122028940 ACN 122028940ACN-122028940-A

Abstract

A PI4K inhibitor and application thereof, in particular to application in blocking or inhibiting autophagy caused by RAS mutation or treating RAS mutation tumor. In addition, it is revealed that autophagy caused by RAS mutation is completely different from that caused by existing starvation induced autophagy in mechanism, the pathway of autophagy caused by RAS mutation is P38-ULK1-PI4KB-WIPI2, especially S256 and T263 sites of ULK1 phosphorylated P14KB, and the specificity of the pathway can be used as a diagnostic marker and a therapeutic target of tumor through experiments.

Inventors

  • GE LIANG
  • ZHANG MIN
  • WANG XIAOJUAN
  • LI SHULIN

Assignees

  • 清华大学

Dates

Publication Date
20260512
Application Date
20240807

Claims (20)

  1. The use of a PI4K inhibitor in the manufacture of a medicament for the prevention and/or treatment of a tumor, wherein the PI4K inhibitor blocks or inhibits PI4K phosphorylation, preferably wherein the tumor is a RAS mutant tumor.
  2. The use according to claim 1, wherein the PI4K inhibitor is used for preventing and/or treating tumors by blocking or inhibiting autophagy caused by RAS mutation.
  3. The use according to claim 1, wherein the PI4K inhibitor prevents and/or treats a tumor by blocking or reducing PI4P levels; Preferably, prevention and/or treatment of tumors is performed by blocking or reducing PI4P levels, thereby blocking or reducing their recruitment interacting proteins; Preferably, the number of species or expression level of PI4P recruited interacting proteins is blocked or reduced.
  4. The use according to claim 3, wherein said interacting protein comprises WD repeat domain phosphoinositide interacting protein 2 (WIPI 2).
  5. The use according to claim 1, wherein the PI4K inhibitor is used for preventing and/or treating tumors by blocking or inhibiting PI4K phosphorylation induced by ULK1 phosphorylation.
  6. The use according to claim 1, wherein the PI4K inhibitor prevents and/or treats tumors by blocking or inhibiting PI4K phosphorylation induced by ULK1 phosphorylation, thereby blocking or reducing PI4P levels, blocking or reducing levels of recruitment interacting proteins, and thus blocking or inhibiting autophagy caused by RAS mutation.
  7. The use of claim 5 or 6, wherein ULK1 phosphorylation comprises ULK1 phosphorylation by the P38 pathway.
  8. The use according to any one of claims 1-7, wherein PI4K is PI4K complex or PI4KB.
  9. The use according to claim 8, wherein the site of PI4KB phosphorylation comprises S256 and/or T263.
  10. The use according to any one of claims 5 to 9, wherein the sites of ULK1 phosphorylation comprise one or more of S317, S556, S758 or S479.
  11. The use according to any one of claims 1-10, wherein the RAS mutation is one or more of a HRAS mutation, a KRAS mutation or a NRAS mutation, preferably a KRAS mutation, such as one or more of G12V, G12C, G12D, G12S, G12R, G13D, G C, A18D, A D, A59T, Q61H, Q61K, Q61L, Q61R, E62G, A146T, K N.
  12. The use according to claim 1 or 11, wherein the tumour is selected from colorectal cancer, pancreatic cancer, cholangiocarcinoma, lung cancer (e.g. non-small cell lung cancer), ovarian cancer, thyroid cancer, bladder cancer, breast cancer, liver cancer, melanoma, myelodysplastic syndrome, lymphoma, endometrial cancer, oesophageal cancer, glioma, squamous cell carcinoma of the head and neck, urothelial cancer, neuroblastoma, renal cancer, leukaemia or multiple myeloma; Preferably, the tumor is colon, lung or pancreatic cancer with RAS mutations.
  13. The use according to any one of claims 1 to 12, wherein the PI4K inhibitor comprises an agent required for gene knockout, an agent required for gene silencing, an agent required for gene mutation, an antisense nucleic acid, a small molecule compound or a pharmaceutically acceptable salt thereof, a polypeptide or an expression enhancer thereof, a polypeptide mutant or an expression enhancer thereof, a fusion protein, an antibody, a traditional Chinese medicine or a traditional Chinese medicine extract.
  14. The use of claim 13, wherein the gene knockout comprises CRISPR or tissue specific knockout.
  15. The use of claim 13, wherein the agent required for gene silencing comprises one or more of interfering RNA, such as siRNA, dsRNA, shRNA, aiRNA or miRNA.
  16. The use of claim 15, wherein the siRNA targets one, two or three of PI4KB, ULK1 or WIPI 2; preferably, the target site sequence of the siRNA targeting ULK1 comprises SEQ ID NO. 2 and/or SEQ ID NO. 3; Preferably, the target site sequence of the siRNA targeting PI4KB comprises one or more than two of SEQ ID NO. 25, SEQ ID NO. 26 or SEQ ID NO. 27; Preferably, the target site sequence of the WIPI2 targeting siRNA comprises one or more of SEQ ID NO. 14, SEQ ID NO. 15 or SEQ ID NO. 16.
  17. The use according to claim 15, wherein the shRNA targets PI4KB and/or ULK1; Preferably, the target site sequence of the shRNA targeting ULK1 comprises SEQ ID NO. 29 and/or SEQ ID NO. 30; Preferably, the target site sequence of the shRNA targeting PI4KB comprises one or more than two of SEQ ID NO:37 and/or SEQ ID NO: 38.
  18. The use according to claim 13, wherein the polypeptide is a polypeptide that is competitively phosphorylated with PI4K, such as an amino acid fragment of PI4KB comprising S256 and/or T263 sites; preferably, the length of the polypeptide is at least 8aa, preferably 8-60aa, further preferably 8-30aa; it is further preferred that the polypeptide comprises at least amino acid sequence positions 256 to 263 of the amino acid sequence of PI4KB, preferably comprising any one of SEQ ID NOS.1 or 83 to 88 or comprising an amino acid sequence having more than 80% identity to any one of SEQ ID NOS.1 or 83 to 88 or comprising an amino acid sequence having up to 10 amino acid substitutions, deletions or insertions to any one of SEQ ID NOS.1 or 83 to 88.
  19. The use according to claim 13, wherein the polypeptide mutant comprises an amino acid fragment of PI4KB with one or more of positions S256, S258, T263 or S266 mutated to non-threonine and non-serine, e.g. mutated to alanine or a non-natural amino acid; Further preferred mutants of the polypeptide include mutants in which serine and threonine at positions 256 to 266 are mutated to alanine, for example any of SEQ ID NOs 1 or 83-88.
  20. Use according to claim 13, wherein the fusion protein comprises a polypeptide which is phosphorylated competitively to PI4K, preferably further comprising an N-terminal Tat protein transduction domain, further preferably the polypeptide is linked to the N-terminal Tat protein transduction domain by GG, e.g. the fusion protein may comprise SEQ ID No. 57 or an amino acid sequence which has more than 80% identity to SEQ ID No. 57 or an amino acid sequence which has up to 10 amino acids substitutions, deletions or insertions to SEQ ID No. 57.

Description

PI4K inhibitor and application thereof Technical Field The invention relates to the technical field of biological medicines, in particular to a P38-ULK1-PI4KB-WIPI2 channel inhibitor and application thereof in blocking or inhibiting autophagy caused by RAS mutation or treating RAS mutation tumor. Background Megaautophagy (hereinafter autophagy) is a conserved degradative metabolic pathway in eukaryotic cells that is critical for the maintenance of intracellular homeostasis and survival of cells under stress conditions. During autophagy, damaged organelles, invasive bacteria and proteins prone to aggregation are degraded by the lysosomal pathway. Autophagy is associated with the pathogenesis of a variety of diseases, for example, autophagy disorders are associated with cancer. However, the specific molecular mechanisms leading to dysregulation of autophagy, particularly the differences between cancer-related autophagy and physiological autophagy (e.g., starvation-induced autophagy), have not been elucidated. The RAS gene family, including HRAS, KRAS and NRAS, encodes very similar 188-189 amino acid proteins, the most common oncogene in human cancers. The RAS gene encodes monomeric gtpase, which functions as a molecular switch in signal transduction pathways that regulate mammalian cell proliferation, differentiation, and survival. Mutations that constitutively activate RAS occur in 20-25% of human cancers, with KRAS mutations occurring most frequently, and in multiple cancer types. In many tumors, activation of KRAS mutations is a key driver of cancer initiation and progression, and is critical to tumor growth. However, highly active RAS mutants are difficult to target due to the lack of drug binding pockets on the RAS protein surface. To date, the only potent inhibitors of clinical use are AMG510 and MRTX849, which specifically target KRAS G12C. In addition to direct targeting of RAS proteins, identifying drugs that inhibit downstream RAS effector proteins is one solution to treat cancers with RAS mutations. Studies have shown that activating RAS mutations are associated with excessive activation of autophagy. RAS-activated autophagy has unique features such as providing nutrition for tumor growth, immune evasion, and remodeling of the proteome by selective degradation, such as elimination of deleterious inflammatory response pathway components to prevent cytokine-induced paracrine cell death. Thus, RAS mutated tumor cells are very sensitive to autophagy inhibitors, indicating that autophagy can be a therapeutic target for RAS mutated tumors. Since autophagy can maintain cell homeostasis under physiological conditions, an autophagy inhibitor should be developed that specifically prevents RAS-induced autophagy without affecting physiological autophagy. However, the regulatory differences between RAS mutation-induced autophagy and physiological autophagy are not yet clear, particularly with respect to the different involvement of the core autophagy factor, the autophagy-related gene (ATG), and the protein. The core steps of autophagy, including the biogenesis and maturation of autophagosomes, are regulated by ATG and proteins. Under starvation-induced autophagy, a physiological form of autophagy, components of the UNC-51-like kinase (ULK) complex, including ULK1/2, ATG13 and Rb 1-inducible frizzled 1 (Rb 1CC1/FIP 200) and ATG101, are activated by inhibiting the mechanical target of rapamycin complex 1 (mTORC 1) or activating AMPK. Downstream of the ULK complex is a class III phosphatidylinositol 3 kinase (PI 3K) complex consisting of Beclin1, ATG14, phosphatidylinositol 3-kinase (PIK 3) catalytic subunit 3 (PIK 3C3/VPS 34), PIK3 regulatory subunit 4 (PIK 3R 4/P150) and activating molecules in Beclin-1 regulated autophagy protein 1. This secondary complex phosphorylates phagocytic Phosphatidylinositol (PI) lipids to recruit phosphatidylinositol-3-phosphate (PI 3P) interacting proteins, such as WD repeat domain phosphoinositide interaction 2 (WIPI 2). WIPI2 is an effector of PI3P, which activates downstream events by recruiting lipid factors (ATG 5-ATG12 conjugates forming complexes with ATG16, ATG3 and ATG 7). The esterification mechanism catalyzes the binding of microtubule-associated protein 1 light chain 3 (MAP 1LC3/LC3/ATG 8) to Phosphatidylethanolamine (PE) to construct autophagosomes. ATG9 was used as a seed for autophagosomes, and together with ATG2 as a lipid turnover enzyme (Scramblase), lipid was transferred to autophagy membranes. ESCRT (endosomal sorting complex required for transport) complexes block the phagosome and thus complete the biogenesis of the autophagosome, multiple SNARE complexes together with membrane tether components promote autophagosome fusion with lysosomes. The cascade of signals and functions in physiological autophagy by ATGs has been largely elucidated, but little is known about how ATGs is regulated under pathological conditions. Disclosure of Invention To make up for the d