Use of a kpna2 agonist and combinations with other agents

Abstract

The application provides use of a specific KPNA2 agonist and a combination of the specific KPNA2 agonist and other substances in preparation of a product for preventing or treating fatty liver. It is found for the first time that the specific KPNA2 agonist can significantly reduce liver triglyceride levels, thereby reducing alcohol-induced liver steatosis, and has good clinical conversion prospects, and the combination of the specific KPNA2 agonist and other substances has better effects.

Description

Uses of a KPNA2 agonist and its combination with other substances Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of a KPNA2 agonist and its combination with other substances. Background Technology

[0002] Currently, there are no effective targeted therapies for fatty liver, such as alcoholic fatty liver disease (ALD), and clinical treatment mainly focuses on alcohol abstinence and supportive care. PPARα is a key nuclear receptor regulating fatty acid β-oxidation, but its nuclear transport in ALD is impaired, affecting transcriptional activity and leading to lipid accumulation. Existing PPARα agonists, such as fenofibrate, can partially improve lipid metabolism, but their effects depend on intact nuclear transport processes, making it difficult to overcome the treatment bottleneck caused by nuclear transport impairment.

[0003] KPNA2 (the α2 subunit of nuclear transporter) is a key member of the nuclear transporter family. Its core function is to mediate the entry of proteins (such as transcription factors and signaling molecules) into the cell nucleus, regulating key processes such as gene expression, proliferation, and apoptosis within the cell. KPNA2 is highly expressed in various cancers and is associated with cancer invasiveness and poor patient prognosis. Related research mainly focuses on how to inhibit KPNA2 function, such as interfering with its mediated nuclear transport through specific binding of nucleic acid aptamers to KPNA2, or using inhibitors to target the binding sites of KPNA2 with other proteins, such as delanzomib targeting the S100A2 / KPNA2 binding site to treat colorectal cancer.

[0004] Agonists are substances in pharmacology and biology that activate specific biomolecules (such as receptors, enzymes, transport proteins, etc.) and trigger their normal physiological functions or enhance their activity. These substances can be small molecule compounds, peptides, hormones, neurotransmitters, etc. Their core function is to "mimic the natural activation signal of the target molecule" or "enhance its function." The core logic of agonists is "precise matching + function activation," which can be understood using the analogy of a key and a lock: The target biomolecule (such as a "receptor") is like a "lock," with a specific "active site" (keyhole) on its structure to receive signals; the agonist is like a "matching key" that precisely binds to the "keyhole" (active site), thus "turning the lock"—that is, activating the molecule's inherent function, allowing it to perform normal physiological tasks (such as signal transmission, catalytic reactions, and substance transport). Agonists are a class of substances that "help target biomolecules 'do their job'." They activate the inherent function of biomolecules through precise binding, either by supplementing the body's natural activation signals (such as using insulin agonists when insulin is deficient) or by enhancing their function to cope with diseases (such as using bronchodilators to activate receptors to relieve asthma). They are important tools in drug development and physiological function regulation.

[0005] There is currently limited research on KPNA2 agonists, and no studies on their relationship with fatty liver have been reported. Summary of the Invention

[0006] To address the aforementioned problems, this invention utilizes the use of a KPNA2 agonist and its combination with other substances, particularly demonstrating its therapeutic effects on fatty liver, especially alcoholic fatty liver.

[0007] Specifically, this application discloses the use of a KPNA2 agonist in the preparation of products for the prevention or treatment of fatty liver.

[0008] A KPNA2 agonist, characterized in that: the KPNA2 agonist comprises a dihydroquinoline derivative, wherein the structure of the dihydroquinoline derivative is as follows:

[0009] , where R represents a C1-C4 alkyl group and X represents a halogen.

[0010] Furthermore, the KPNA2 agonist includes a dihydroquinoline derivative, the structure of which is as follows:

[0011] , where R represents a C1-C4 alkyl group and X represents a halogen.

[0012] The products include pharmaceuticals.

[0013] Furthermore, the fatty liver is selected from one of alcoholic fatty liver, non-alcoholic fatty liver, malnutrition-related fatty liver, and drug-induced fatty liver.

[0014] Furthermore, the fatty liver mentioned is alcoholic fatty liver.

[0015] Furthermore, in the structural formula of the dihydroquinoline derivative, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, or isobutyl; and X is F or Cl.

[0016] Furthermore, the structure of the dihydroquinoline derivative is as follows: (Hereinafter referred to as Fos).

[0017] Furthermore, the product further comprises a PPARα agonist.

[0018] Furthermore, the PPARα agonist is selected from one or more of fenofibrate, gemfibrozil, sitagliptazakate, bezafibrate, ciprofibrate, clofibrate, aleglitazar, and elafibranor.

[0019] Furthermore, the dosage of the KPNA2 agonist is 1-20 mg / kg / day, preferably 10 mg / kg / day; the dosage of the PPARα agonist is 20-80 mg / kg / day, preferably 40-50 mg / kg / day. The dosage of dihydroquinoline derivatives, especially Fos, is 1-20 mg / kg / day, preferably 10 mg / kg / day, and the dosage of Fenofibrate is 20-80 mg / kg / day, preferably 40-50 mg / kg / day.

[0020] This invention is the first to discover that KPNA2 agonists, especially dihydroquinoline derivatives, significantly reduce liver triglyceride levels, thereby alleviating alcohol-induced hepatic steatosis. This has good prospects for clinical translation, and it can also be used in combination with PPARα agonists to achieve better results. Attached Figure Description

[0021] Figure 1 is a two-dimensional molecular docking interaction diagram of Fos-mediated KPNA2.

[0022] Figure 2 shows the KPNA2 immunoblotting. HepG2 cells were treated with different concentrations of Foslinanib (0, 10, 50, 100, 500 nM) for 24 h, and the KPNA2 protein level was detected.

[0023] Figure 3. Liver tissues of WT and Kif13b KO mice were paraffin-embedded and stained with hematoxylin and eosin (HE) after alcohol treatment.

[0024] Figure 4a. Frozen sections of liver tissue from WT and Kif13b KO mice stained with Oil Red O.

[0025] Figure 4b is a bar chart showing the statistical results of the positive area of ​​Oil Red O in Figure 4a.

[0026] Figure 5a shows the detection of lipid droplet deposition in liver tissues of WT and KO mice after BODIPY staining (green represents lipid droplets, blue represents cell nuclei).

[0027] Figure 5b is a bar chart showing the statistical results of the fluorescent positive area in Figure 5a.

[0028] Figure 6. Triglyceride (TG) levels in liver tissue of WT and KIF13B-KO mice after alcohol treatment. Detailed Implementation

[0029] Figure 1 shows a two-dimensional docking plot of Fos and KPNA2. This docking plot reveals that Fos binds stably to KPNA2 through a combination of hydrophobic pocket intercalation, hydrogen bonding, and water molecule bridging, providing a structural basis for its role as a KPNA2 agonist.

[0030] Specifically, the realization of KPNA2 agonists involves the following five factors:

[0031] 1. Molecular skeleton and functional groups

[0032] Central structure: The core of Fos is a polycyclic aromatic structure (similar to quinoline or indole derivatives) with a phosphate group (–PO3). 2- ), and a fluorinated aromatic ring.

[0033] Functional groups:

[0034] Phosphate group (left): highly polar, often binds to hydrogen bond donors / acceptors or water molecules.

[0035] Aromatic ring + fluorine (right side): Increased hydrophobicity, which facilitates entry into the hydrophobic pocket and the formation of hydrophobic interactions with the amino acid side chain.

[0036] Carbonyl group & NH: provide hydrogen bond donor / acceptor functions, which facilitates stable binding.

[0037] 2. Key amino acid interactions

[0038] HIS418, VAL417, VAL414, ILE413, ILE447: surround the bottom to form a hydrophobic pocket, stabilizing the aromatic skeleton of the drug.

[0039] ALA451, LEU454, and LEU460: also provide hydrophobic interactions, enhancing drug binding stability.

[0040] GLU456: It forms hydrogen bonds with NH molecules (see purple arrow), which is an important polar interaction site.

[0041] LYS459: Charge interactions or electrostatic repulsion may form nearby, but this figure shows possible neighboring interaction sites.

[0042] 3. Phosphate groups and water molecules

[0043] Phosphate group (–PO3) 2- The figure shows that it forms hydrogen bond bridging with two water molecules (H2O) (see pink arrow on the left).

[0044] This indicates that the group helps to enhance ligand-protein binding affinity through polar interactions.

[0045] 4. Summary of Interaction Modes

[0046] Hydrophobic interactions: Extensive hydrophobic interactions between aromatic rings and hydrophobic amino acids (VAL, ILE, LEU, ALA) constitute the main stabilizing forces.

[0047] Hydrogen bonding: The hydrogen bond between NH in the molecule and GLU456 plays a key positioning role; the phosphate group forms a hydrogen bond network with water molecules, which enhances the binding.

[0048] Fluorine-substituted aromatic rings: Fluorine atoms enhance hydrophobicity and electronic effects, improving the fit with hydrophobic pockets.

[0049] 5. Biological significance

[0050] This indicates that Fos can stably bind within the binding pocket of KPNA2 through multi-point interactions (hydrophobicity + hydrogen bonding). In particular, the hydrogen bonding at GLU456 and the hydrophobic pocket (VAL / ILE / LEU / ALA) are the main contributing factors to this binding. After binding, Fos is expected to stabilize the KPNA2 protein structure, enhance its nuclear transport function, thereby promoting the nuclear translocation of key factors such as PPARα and restoring lipid metabolism regulation.

[0051] Based on the above description, meeting some of the requirements could potentially make an agent for KPNA2, and it is not limited to dihydroquinoline derivatives or Fos as described in this application. The above is only an example of how to achieve a KPNA2 agonist using Fos.

[0052] The following experiments verified the effects of KPNA2 agonists on fatty liver, especially alcoholic fatty liver.

[0053] This application uses two types of mice: WT (Wild-Type): wild-type C57BL / 6J mice with complete genotype and normal KIF13B expression.

[0054] KO (Knockout): refers to KIF13B gene knockout mice, in which KIF13B function is lost in the liver, used to verify the key role of this gene in disease development and drug action.

[0055] WT (wild-type mice) represents a physiological state in which KIF13B functions normally. In this context, KIF13B can stabilize KPNA2 and maintain nuclear transport of PPARα, thereby ensuring the normal progress of fatty acid β-oxidation.

[0056] KO (KIF13B knockout mouse) mice mimic the pathological state of KIF13B dysfunction. In KO mice, although the protein expression level of PPARα is normal, the lack of KIF13B leads to decreased stability of KPNA2, and PPARα cannot effectively translocate into the cell nucleus, thus resulting in a state of "functional inactivation".

[0057] In this situation, even with the administration of PPARα agonists (such as fenofibrate), their transcriptional activation capacity is significantly weakened because PPARα cannot enter the nucleus, resulting in limited therapeutic efficacy. Based on this finding, for patients with similar KIF13B functional deficiencies or nuclear transport disorders, the use of PPARα agonists alone may not be sufficient to restore lipid metabolism function.

[0058] Therefore, this patent proposes a new combined treatment approach: in the context of KIF13B deficiency or ALD and other impaired nuclear transport, a combination of KPNA2 agonists (such as Fos, used to restore PPARα nuclear translocation) and PPARα agonists (such as fenofibrate, used to activate its transcriptional function) is used. Through upstream and downstream dual intervention, not only can the nuclear localization of PPARα be restored, but its downstream metabolic pathways can also be fully activated, thereby achieving a more significant therapeutic effect.

[0059] In other words, the choice of WT and KO as experimental controls was precisely to demonstrate that KIF13B deficiency → PPARα nuclear entry barrier → weakened efficacy of PPARα agonists, thus highlighting the clinical necessity and scientific basis of combined treatment with KPNA2 agonists and PPARα agonists.

[0060] The relationship between KIF13B, KO and KPNA2

[0061] In WT mice: KIF13B is present → it can bind to KPNA2 and stabilize its protein level.

[0062] Stable KPNA2 effectively promotes PPARα entry into the nucleus, driving the fatty acid β-oxidation transcriptional program.

[0063] In KO mice: Lack of KIF13B → decreased KPNA2 protein stability. Insufficient KPNA2 levels → PPARα mainly remains in the cytoplasm, leading to impaired β-oxidation and accumulation of lipid droplets in the liver.

[0064] The drug Fos, as a KPNA2 agonist, can promote PPARα nuclear translocation by directly stabilizing KPNA2, even in the absence of KIF13B, thereby partially compensating for the defects caused by KO.

[0065] Example 1:

[0066] Experimental methods and grouping: Eight-week-old C57BL / 6J wild-type female mice (WT) and Kif13bKO (KO) female mice were randomly divided into two groups. After two weeks of modeling with a Lieber-DeCarli alcoholic liquid diet, the mice were treated with solvent (control) and Fos (10 mg / kg / day, Fos) for two weeks.

[0067] Figure 2. Western blot (WB) demonstrates that Foslinanib upregulates KPNA2 protein levels in a dose-dependent manner, indicating that Foslinanib is a KPNA2 agonist.

[0068] Figure 2 shows that Foslinanib upregulates KPNA2 protein levels in a dose-dependent manner, demonstrating that it is an agonist of KPNA2.

[0069] Experimental subjects: HepG2 cells (a type of hepatocyte) were treated with different concentrations of Foslinanib (0, 10, 50, 100, 500 nM), and the KPNA2 protein level was detected.

[0070] Results: Western blotting showed that KPNA2 protein expression gradually increased with increasing Foslinanib concentration. The internal control GAPDH band remained stable, indicating consistent protein loading. Quantitative analysis (right figure) showed that compared with the control group (0 nM), Foslinanib significantly increased KPNA2 protein levels at 50 nM, 100 nM, and 500 nM in a dose-dependent manner. Statistical tests (P < 0.05 to P < 0.001) further validated the significant effect of Foslinanib on improving KPNA2 stability.

[0071] Figure 3 (HE staining, liver histopathology)

[0072] Figure 3 shows HE staining, which indicates that Foslinanib can significantly improve alcoholic liver injury, and produces a stronger protective effect when used in combination with Fenofibrate, suggesting that combined intervention of upstream and downstream dual targets is an effective treatment strategy.

[0073] HE staining results showed that alcohol treatment resulted in disordered hepatocyte arrangement, steatosis, and inflammatory infiltration in WT mice, while KO mice exhibited more severe pathological damage. Fos treatment significantly improved liver histopathology in both WT and KO mice, with similar degrees of lesion severity, suggesting that Fos can effectively alleviate alcoholic liver injury, and its pathological protective effect is similar in both WT and KO cases. The combined use of Foslinanib and Fenofibrate produced a stronger protective effect, revealing a potential therapeutic strategy involving combined upstream and downstream target intervention.

[0074] Figure 4 (a shows Oil Red O staining, b shows the positive area of ​​Oil Red O staining)

[0075] Figure 4a shows Oil Red O staining, which indicates that KIF13B deficiency exacerbates alcoholic liver lipid droplet deposition, while Foslinanib can significantly improve this lesion, and the combined treatment with Fenofibrate has the strongest effect.

[0076] Figure 4b is a bar chart showing the statistical results of the positive area of ​​Oil Red O in Figure 4a.

[0077] Figure a (Oil Red O staining). WT Control: After alcohol treatment, numerous red lipid droplets were deposited in the liver tissue. WT Fos: Foslinanib significantly reduced red lipid droplets, and liver lipid accumulation was significantly improved. WT Fos+FB: Lipid droplets were further reduced, almost returning to near-normal levels. KO Control: Compared with WT, KO mice showed more severe lipid droplet deposition, indicating that KIF13B deficiency exacerbates alcoholic liver lipid accumulation. KO Fos: Foslinanib significantly reduced lipid droplets, but the improvement was slightly less than that in WT. KO Fos+FB: The reduction in lipid droplets was most significant, suggesting that combined medication has a stronger protective effect.

[0078] Figure b (quantification of Oil Red O positive area). Both the WT and KO control groups showed high levels of lipid droplet area, with KO showing higher areas than WT. Foslinanib significantly reduced the lipid droplet area in both groups of mice (P < 0.0001). The Fos+FB group had the lowest lipid droplet area, which was significantly lower than that of the Fos monotherapy group.

[0079] Figure 5 (a) shows BODIPY staining, with green indicating lipid droplets; b) shows the statistical results of the fluorescent positive area.

[0080] Figure 5a shows BODIPY staining, indicating that KIF13B deficiency exacerbates alcoholic liver lipid droplet deposition, while Foslinanib significantly reduces lipid droplet levels in both WT and KO mice, and its effect depends on activating KPNA2 to promote PPARα nuclear translocation.

[0081] Figure 5b is a bar chart showing the statistical results of the fluorescent positive area in Figure 5a.

[0082] Figure a (BODIPY staining, green represents lipid droplets, blue represents cell nuclei). WT Control: After alcohol treatment, a significant green fluorescent signal is visible in hepatocytes, representing lipid droplet deposition. WT Fos: Foslinanib treatment significantly reduces green fluorescence, indicating a significant decrease in lipid droplets. KO Control: Compared to WT, KIF13B deficiency leads to a stronger green fluorescent signal and more severe lipid droplet accumulation. KO Fos: Foslinanib treatment significantly reduces the intensity of green fluorescence, bringing lipid droplet levels close to the WT Fos group.

[0083] Figure b (Quantitative BODIPY Statistical Analysis). Both the WT and KO control groups showed significantly enhanced lipid droplet fluorescence, with the KO group showing the highest level, indicating that KIF13B deficiency exacerbates alcoholic lipid droplet deposition. The Fos-treated groups (WT and KO) significantly reduced BODIPY fluorescence intensity (P < 0.01–0.0001), suggesting that Foslinanib can effectively reduce lipid droplet accumulation. The KO group showed a significant decrease in lipid droplet levels after Fos treatment, almost returning to the levels of the WT Fos group.

[0084] KIF13B deficiency exacerbated alcohol-induced hepatic lipid droplet deposition, demonstrating its protective role in maintaining lipid homeostasis. Foslinanib significantly reduced hepatic lipid droplet levels in both WT and KO mice, thereby improving alcoholic lipid accumulation. Foslinanib remained effective in KO mice, suggesting that its mechanism of action primarily involves activating KPNA2 and promoting PPARα nuclear transport.

[0085] Figure 6 (Quantitative detection of triglycerides in liver tissue)

[0086] Figure 6 shows the quantitative analysis of TG in liver tissue, which indicates that KIF13B deficiency significantly aggravates alcoholic liver triglyceride deposition, while Foslinanib, especially when used in combination with Fenofibrate, can significantly reduce TG levels in WT and KO mice, demonstrating the advantages of combined intervention.

[0087] KO mice had higher TG levels than WT mice, indicating that KIF13B deficiency further exacerbated alcohol-induced hepatic triglyceride deposition. Foslinanib treatment significantly reduced TG levels in both WT and KO mice (P < 0.001), demonstrating its significant protective effect against alcohol-induced lipid accumulation. The Fos+Fenofibrate combined treatment group showed a further decrease in TG levels, reaching the lowest levels in both WT and KO mice, suggesting a synergistic effect of the combined treatment. This indicates that KIF13B deficiency significantly aggravates alcohol-induced hepatic triglyceride deposition, further confirming its protective role in maintaining lipid metabolism. Foslinanib significantly reduced hepatic TG levels, remaining effective even in the context of KO. The combined treatment (Fos+Fenofibrate) showed the best effect, demonstrating that simultaneous intervention in nuclear transport (Foslinanib→KPNA2) and receptor activation (Fenofibrate→PPARα) produces a stronger therapeutic effect. The figure demonstrates that KIF13B deficiency exacerbates alcoholic liver TG deposition, while Foslinanib, especially when used in combination with Fenofibrate, significantly reduces liver triglyceride levels in WT and KO mice, revealing the superiority of the combined intervention strategy.

[0088] Explanation of symbols in the diagram

[0089] p > 0.05 is considered to be statistically insignificant; p < 0.05 is considered to be statistically significant.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a KPNA2 agonist, characterized in that: The intended use is in the preparation of products for the prevention or treatment of fatty liver, wherein the KPNA2 agonist comprises a dihydroquinoline derivative, the structure of which is... 。 2. The use according to claim 1, characterized in that: The fatty liver is selected from one of the following: alcoholic fatty liver, non-alcoholic fatty liver, malnutrition-induced fatty liver, and drug-induced fatty liver.

3. The use according to claim 1, characterized in that: The fatty liver mentioned is alcoholic fatty liver.

4. The use according to claim 1, characterized in that: The product further comprises a PPARα agonist selected from fenofibrate.

5. The use according to any one of claims 1-4, characterized in that: The dosage of KPNA2 agonists is 1-20 mg / kg / day; the dosage of PPARα agonists is 20-80 mg / kg / day.

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