Application of RYK and ligand-receptor signal axis inhibitor thereof in preparation of medicine for treating fatty liver disease and pulmonary fibrosis

Abstract

The invention discloses application of RYK and a ligand-receptor signal axis inhibitor thereof in preparation of medicines for treating fatty liver diseases and pulmonary fibrosis, in the technical scheme provided by the invention, the RYK receptor is found and verified as a functional receptor of a secretory extracellular domain (G-ECD) of glycoprotein non-metastatic melanoma protein B for the first time; it is revealed that a G-ECD-RYK signal axis is a key mechanism for driving occurrence and development of metabolic dysfunction related fatty liver diseases / hepatitis (MASLD / MASH) and pulmonary fibrosis. According to the invention, the ligand G-ECD of the RYK is found, and the ligand-receptor signal axis of the RYK is determined to be a brand new drug target with high specificity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of RYK and its ligand-receptor signaling axis inhibitors in the preparation of drugs for treating fatty liver disease and pulmonary fibrosis. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease / steatohepatitis (MASLD / MASH) has become the most common chronic liver disease worldwide, with its prevalence rising sharply due to the prevalence of obesity and metabolic syndrome. Currently, a significant number of adults globally are affected by this disease. The spectrum of MASLD / MASH progresses from simple steatosis to steatohepatitis, liver fibrosis, and ultimately cirrhosis and hepatocellular carcinoma. Despite this disease being a serious public health concern, the number of effective drugs approved by the FDA or other regulatory agencies is far from sufficient. Existing treatments include resmetirom, semaglutide, and lifestyle interventions (dietary control and exercise), but their efficacy is limited and patient adherence is poor. Although several drugs (such as obeticholic acid, liraglutide, and vitamin E) have entered clinical trials in recent years, they all suffer from insufficient efficacy, significant side effects, or unclear long-term safety. More importantly, existing therapeutic targets (such as FXR and PPAR) mainly target metabolic regulation and inflammatory responses, with limited effectiveness in reversing liver fibrosis, a key driver of disease progression. Therefore, there is an urgent need to discover new molecular targets and pathogenic mechanisms to develop innovative therapies that can simultaneously improve steatosis, inflammation, and fibrosis.

[0003] Pulmonary fibrosis is a late-stage manifestation of interstitial lung disease characterized by fibroblast proliferation and extensive extracellular matrix deposition, leading to the destruction of lung tissue structure. A common type of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF), which primarily affects middle-aged and elderly individuals, but its specific etiology remains unclear. Factors contributing to pulmonary fibrosis are diverse, including occupational or home environment factors, medications, radiation therapy, high-concentration oxygen therapy, smoking, disease-related factors, and genetic factors. Currently, only three drugs—nintedanib, nerandomilast, and pirfenidone—are approved for the treatment of IPF, but their effectiveness in reversing existing fibrosis is limited. Furthermore, the side effects of these drugs (such as gastrointestinal reactions, abnormal liver function, and photosensitivity) severely impact patients' quality of life and medication adherence. More importantly, existing drugs target too broadly (e.g., the TGF-β signaling pathway, tyrosine kinases), lacking tissue specificity, resulting in a narrow therapeutic window. For pulmonary fibrosis caused by different etiologies (such as radiation-induced lung injury, drug-induced pulmonary fibrosis, and fibrosis related to the sequelae of COVID-19 infection), there are currently no effective treatments. Therefore, in-depth exploration of the common molecular mechanisms of the occurrence and development of pulmonary fibrosis, discovery of new specific therapeutic targets, and development of broad-spectrum therapeutic drugs applicable to pulmonary fibrosis of multiple etiologies have significant clinical and social value. Summary of the Invention

[0004] The main objective of this invention is to propose the application of receptor-like tyrosine kinase (RYK) and its ligand-receptor signaling axis inhibitors in the preparation of drugs for the treatment of fatty liver disease and pulmonary fibrosis. The aim is to elucidate the key pathogenic role of RYK receptors and their ligand signaling axes in the development of MASH and pulmonary fibrosis, reveal a new ligand-receptor signaling pathway, and provide novel molecular targets with potential translational value for the targeted prevention and treatment of MASH and pulmonary fibrosis, so as to develop innovative therapies that can simultaneously prevent or treat MASH and pulmonary fibrosis.

[0005] To achieve the above objectives, this invention proposes the application of RYK and its ligand-receptor signaling axis inhibitors in the preparation of drugs for treating fatty liver disease and pulmonary fibrosis.

[0006] Preferably, the fatty liver disease is MASLD or MASH, and the pulmonary fibrosis includes idiopathic pulmonary fibrosis, environmentally induced pulmonary fibrosis, drug-induced pulmonary fibrosis, post-infectious pulmonary fibrosis (including post-COVID-19 infection), and other disease-related pulmonary fibrosis.

[0007] Preferably, the ligand is a glycoprotein nonmetastatic melanoma protein B or its secretory extracellular domain G-ECD.

[0008] Preferably, the inhibitor blocks the interaction between the glycoprotein nonmetastatic melanoma protein B and its receptor RYK.

[0009] Preferably, the inhibitor specifically blocks the binding between G-ECD and the WIF domain of the RYK extracellular domain.

[0010] Preferably, the inhibitor reduces the expression level of RYK or glycoprotein nonmetastatic melanoma protein B.

[0011] Preferably, the inhibitor includes one or more of the following: vaccine, shRNA, siRNA, neutralizing antibody, small molecule inhibitor, and protein inhibitor.

[0012] The present invention also proposes a pharmaceutical composition comprising an inhibitor targeting RYK or an inhibitor targeting G-ECD.

[0013] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] The present invention also proposes a combination of biomarkers for detecting the severity of progression of MASLD / MASH and / or pulmonary fibrosis, the combination of biomarkers including G-ECD and RYK.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The technical solution provided by this invention discovers and verifies for the first time that the RYK receptor is a functional receptor for the secretory extracellular domain (G-ECD) of glycoprotein nonmetastatic melanoma protein B, revealing that the RYK ligand-receptor signaling axis is a key mechanism driving the development of metabolic dysfunction-associated fatty liver disease / hepatitis (MASLD / MASH) and pulmonary fibrosis. This invention, through step-by-step deletion mutations of the functional segments of the RYK receptor and protein immunoprecipitation, further clarifies that the binding of G-ECD to RYK is strictly dependent on the WIF domain of the RYK extracellular domain. This discovery not only breaks through the limitations of previous understanding of the pathogenic mechanism of glycoprotein nonmetastatic melanoma protein B, but also establishes RYK as a novel and highly specific drug target. Intervention targeting this target can block the pathogenic signal transduction of "ligand-receptor" at the source, thereby overcoming the technical defects of existing targets (such as TGF-β) such as narrow therapeutic window and large side effects due to their wide range of effects. This provides a solid theoretical and material basis for the development of broad-spectrum and highly effective drugs that target both liver and lung fibrosis.

[0016] This invention provides several innovative therapeutic strategies targeting and inhibiting the RYK-G-ECD signaling axis, including vaccine immunization, neutralizing antibodies, siRNA, shRNA, and small molecule or protein inhibitors, and has demonstrated their significant clinical translational potential in animal models. Experimental data show that both blocking the physical binding of G-ECD to RYK through antibodies / vaccines and specifically downregulating target gene expression through siRNA / shRNA can significantly reduce hepatic lipid deposition, alleviate inflammatory infiltration, and reverse the fibrotic process in liver / lung tissues. In particular, GalNAc-siRNA technology achieves hepatocyte-specific delivery, minimizing off-target risks while ensuring efficacy. Furthermore, this invention demonstrates a positive correlation between serum G-ECD levels and disease severity, making it an ideal non-invasive biomarker for assisting disease diagnosis, patient stratification, and efficacy monitoring, greatly enhancing the clinical application value of this technology. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The diagram shows the discovery of the G-ECD-RYK ligand-receptor signaling axis in this invention; A: Schematic diagram of the RYK protein domain; B: Diagram showing the binding of RYK to G-ECD and the verification of domain requirements.

[0019] Figure 2 The diagram shows the effects of this invention's inhibition of RYK and its ligand-receptor signaling axis on fatty liver disease; A: Diagram showing the effects of RYK inhibition on the development of MASH; B: Diagram of a mouse model of hepatocyte-specific RYK knockout / G-ECD overexpression; C: Diagram showing the effects of vaccine immunization, antibody therapy, or siRNA interference with the RYK ligand-receptor signaling axis on the progression of fatty liver disease.

[0020] Figure 3 The diagram shows the effect of the present invention on pulmonary fibrosis by inhibiting RYK and its ligand-receptor signaling axis in lung tissue; A: Diagram showing the improvement of bleomycin-induced pulmonary fibrosis by inhibiting RYK; B: Diagram showing the improvement of COVID-19-induced pulmonary fibrosis by neutralizing antibody inhibiting RYK ligand-receptor binding.

[0021] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0024] Example 1: Discovery of the G-ECD-RYK ligand-receptor signaling axis This study identified the RYK ligand G-ECD, and this ligand-receptor axis is a key pathogenic signaling pathway in metabolic dysfunction-associated steatotic liver disease / steatohepatitis (MASLD / MASH) and pulmonary fibrosis (including COVID-19-induced pulmonary fibrosis). We demonstrated this novel ligand-receptor signaling axis using stepwise deletion mutations in the functional region of the RYK receptor and protein immunoprecipitation, clarifying that its ligand-receptor binding is strictly dependent on the WIF domain of the RYK extracellular domain.

[0025] 1. Experimental Methods G-ECD-RYK interaction and identification of binding domains We cloned the full-length RYK cDNA into the pCDNA3 vector and fused a Myc tag sequence to the C-terminus. Using the ClonExpress II one-step cloning kit, we prepared mutant RYK plasmids in different truncated forms. Then, we cloned the G-ECD region cDNA into the pCMV14 vector and fused a Flag tag sequence to the C-terminus. The G-ECD region (amino acid sequence shown in SEQ ID NO: 2) and mutant RYK plasmids from different species were transfected into Huh7 cells. Forty-eight hours after transfection, the cells were incubated on ice for 30 minutes using non-denaturing lysis buffer to obtain cell lysates. Add anti-Flag affinity gel agarose beads, gently mix on a shaker at 4 ℃ for 1 hour, wash the agarose beads three times with phosphate buffer, mix the protein sample with immunoblotting buffer, heat to 95 ℃ for 10 minutes to denature, separate the protein bands using SDS-PAGE, transfer to a nitrocellulose membrane in transfer buffer, block with skim milk for 1 hour, gently shake and incubate overnight at 4 ℃ with anti-Flag and anti-Myc antibodies (1:1000 dilution), wash with secondary antibody, perform chemiluminescence staining, and detect the RYK mutant that binds to G-ECD-Flag protein to confirm the RYK protein domain that G-ECD protein binds to.

[0026] 2. Experimental Results RYK protein structure analysis and identification of RYK as a G-ECD receptor RYK is a single-transmembrane pseudokinase protein. Its N-terminal extracellular segment (NTD) contains a WNT repressor (WIF) domain, and its C-terminal intracellular segment (CTD) contains a protein tyrosine kinase domain and a tripeptide-binding motif (AYV). Figure 1 A). We found that G-ECD binds to the extracellular domain (ECD) of RYK, and that this binding interaction requires the WIF domain of RYK. Figure 1 B).

[0027] Example 2: Inhibition of RYK and its ligand-receptor signaling axis (vaccines, neutralizing antibodies, hepatocyte-targeted GalNAc-siRNA) can effectively improve fatty liver disease. 1. Experimental Methods (1) Using AAV8-shRNA to knock down RYK expression in mouse liver to induce MASH model Sixteen 8-week-old male wild-type mice were randomly divided into two groups: eight mice served as the AAV8-shNC control group, and eight mice served as the AAV8-shRyk experimental group. Each group received a tail vein injection of 1 × 10⁻⁶ mg / L. 11AAV8-shNC and AAV8-shRyk viral genome copies were used to induce a MASH mouse model with AMLN diet for 6 months. Liver tissue was collected, and gross liver images were taken. All mouse liver tissues were dehydrated, paraffin-embedded, and liver sections were stained with H&E, Oil Red O, α-SMA immunostaining, and Sirius Red staining to detect liver damage, lipid accumulation, and fibrosis. MASLD histological evaluation was performed.

[0028] (2) Construction of hepatocyte-specific RYK knockout / G-ECD overexpression mice and MASH modeling We used a hepatocyte-specific albumin promoter to drive G-ECD overexpression in mouse liver, constructing a transgenic mouse (Alb-G-ECD) that specifically overexpresses G-ECD. By inserting introns 2 and 4 of the Ryk gene (encoding the amino acid sequence shown in SEQ ID NO: 1) into the LoxP site, we constructed Ryk flox (Ryk... fl / fl ) mice, and crossed with Alb-G-ECD mice to obtain Alb-G-ECD; Ryk fl / fl Mouse. Establish Ryk fl / fl AAV8-TBG-NC control group, Alb-G-ECD; Ryk fl / fl AAV8-TBG-NC control group, Ryk fl / fl AAV8-TBG-Cre experimental group, Alb-G-ECD; Ryk fl / fl The AAV8-TBG-Cre experimental groups consisted of 7 mice per group. Hepatocyte-specific Ryk knockout was achieved by transducing Cre recombinase driven by the hepatocyte-specific TBG promoter. The MASH model was induced in mice with AMLN diets for 5 months. Liver tissue was collected, and gross liver images were taken. All mouse liver tissues were dehydrated, paraffin-embedded, and liver sections were stained with H&E and Sirius red to detect liver damage, lipid accumulation, and fibrosis. MASLD histological evaluation was performed.

[0029] (3) Peptide vaccines, neutralizing antibodies, and siRNA inhibit the mouse RYK signaling axis We used an antigenic peptide fragment (60 µg, RRGDGRWKD) to bind to the immunogenic carrier protein KLH and emulsified it with MnJ(β) adjuvant (300 µg). Eight-week-old male wild-type mice were injected with the emulsified peptide vaccine via subcutaneous, intraperitoneal, and intramuscular injections at multiple sites, for a total of four injections. The first two injections were 10 days apart, and the subsequent three injections were 7 days apart. During the vaccination period, they continued to be fed an AMLN diet for a total of 6 months. In the neutralizing antibody and GalNAc-siRNA treatment regimen, eight-week-old male wild-type mice were fed an AMLN diet for 4 months and injected intraperitoneally with neutralizing antibodies (5 mg / kg body weight) twice a week for 2 months; or injected subcutaneously with GalNAc-siRNA (GmsAmsGmAmGfAmGfCfAfCmAmAmCmCmAmAmUmUmAm) (4 mg / kg body weight) once a month for 2 months, while continuing to be fed an AMLN diet. At the end of the experiment, liver tissue was collected, and gross images of the liver were taken. All mouse liver tissues were dehydrated, paraffin-embedded, and liver sections were stained with H&E and Oil Red O to detect liver damage and lipid accumulation.

[0030] 2. Experimental Results The results showed that RYK knockdown mice had reduced hepatic lipid accumulation and alleviated fibrosis, demonstrating that inhibiting RYK effectively improves the occurrence and development of MASH. Figure 2 A).

[0031] To further confirm that RYK is a downstream effector receptor mediating MASH, we constructed a transgenic mouse model that specifically overexpresses G-ECD in hepatocytes (Alb-G-ECD) and, through interaction with Ryk... fl / fl Mouse hybridization and AAV8-TBG-Cre infection were used to obtain a hepatocyte-specific RYK knockout / G-ECD overexpression mouse model. Figure 2 B). A mouse MASH model was induced. Experimental results showed that Alb-G-ECD mice were more sensitive to MASH, demonstrating that G-ECD promotes the MASH process; knockout of RYK alone improved the MASH phenotype; RYK knockout could inhibit G-ECD-mediated MASH pathogenesis. Figure 2 B).

[0032] We evaluated the efficacy of vaccines, neutralizing antibodies, and GalNAc-siRNA targeting the signaling axis of RYK and its ligands in the prevention and treatment of MASH. Vaccines or neutralizing antibodies were prepared using antigenic peptide fragments of RYK and its ligands. An AMLN-induced mouse model of MASH was established, and wild-type mice were vaccinated, administered neutralizing antibodies or siRNA, and then continued to be fed an AMLN diet. Compared with control mice, vaccine immunization, antibody treatment, or siRNA interference significantly improved the progression of fatty liver disease and reduced steatosis (…). Figure 2 C).

[0033] In summary, targeting the RYK receptor-ligand signaling axis is a promising strategy for the prevention and treatment of fatty liver disease.

[0034] Example 3: Inhibiting RYK and its ligand-receptor signaling axis in lung tissue can effectively improve pulmonary fibrosis. 1. Experimental Methods (1) Lung tissue RYK knockout / G-ECD overexpression and bleomycin-induced pulmonary fibrosis model 8-week-old male G-ECD flox (G-ECD fl / fl Fourteen mice were randomly divided into two groups of seven each. Eight-week-old male macrophage-specific G-ECD knockout (G-ECD) mice were used. △MP Eighteen mice were randomly divided into three groups of six each. We studied G-ECD... fl / fl and G-ECD △MP Mice were infected by intratracheal injection of 3 × 10 11 The viral genome copy number was AAV9-shRyk or AAV9-G-ECD. Ten days later, mice were anesthetized and weighed. Bleomycin (BLM, 4 U / kg body weight) was injected intratracheally via a high-pressure nebulizer to establish a mouse model of pulmonary fibrosis. Twenty-one days later, lung tissue was collected from the mice, and whole-lung CT scans were performed to detect lung parenchyma. The mouse lung tissue was dehydrated, paraffin-embedded, and lung sections were stained with H&E and Massen's trichrome to detect lung injury and fibrosis progression.

[0035] (2) SARS-CoV-2-induced pulmonary fibrosis model caused by COVID-19 and the inhibition of RYK signaling axis in mouse lung tissue by neutralizing antibodies Sixteen 15-month-old human ACE2 transgenic mice were randomly divided into two groups of eight each. One × 10⁸ mice were infected via nasal droplets. 5 Half-maximal dose of mouse-adapted SARS-CoV-2 was administered via intranasal droplets of G-ECD neutralizing antibody (2 mg / kg body weight) on days 3 and 7 post-infection. On day 10 post-infection, mouse lungs were collected, and lung tissue sections were stained with H&E and Massen trichrome to detect lung injury and fibrosis progression.

[0036] 2. Experimental Results RYK knockdown or its ligand G-ECD knockout in lung tissue effectively inhibited pulmonary fibrosis; AAV9-G-ECD-mediated G-ECD re-expression eliminated the protective effect of macrophage-specific G-ECD deficiency; RYK knockdown mice remained resistant to G-ECD overexpression-mediated fibrosis progression and significantly improved pulmonary fibrosis progression. Figure 3 A).

[0037] Ten days after SARS-CoV-2 infection, mice developed severe pulmonary fibrosis with significant thickening of the alveolar septa and severe immune cell infiltration. Compared with control mice, neutralizing antibody treatment significantly slowed the progression of fibrosis, reduced fibroblast proliferation, inflammatory infiltration, and collagen deposition. Figure 3 B).

[0038] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. Application of RYK and its ligand-receptor signaling axis inhibitors in the preparation of drugs for the treatment of fatty liver disease and pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The fatty liver disease is MASLD or MASH, and the pulmonary fibrosis includes idiopathic pulmonary fibrosis, environmentally induced pulmonary fibrosis, drug-induced pulmonary fibrosis, post-infection pulmonary fibrosis, and pulmonary fibrosis related to other diseases.

3. The application according to claim 1, characterized in that, The ligand is the glycoprotein nonmetastatic melanoma protein B or its secretory extracellular domain G-ECD.

4. The application according to claim 1, characterized in that, The inhibitor blocks the interaction between the glycoprotein nonmetastatic melanoma protein B and its receptor RYK.

5. The application according to claim 4, characterized in that, The inhibitor specifically blocks the binding between G-ECD and the WIF domain of the RYK extracellular domain.

6. The application according to claim 1, characterized in that, The inhibitor reduces the expression level of RYK or glycoprotein nonmetastatic melanoma protein B.

7. The application according to claim 1, characterized in that, The inhibitors include one or more of the following: vaccines, shRNA, neutralizing antibodies, N-acetylgalactosamine-siRNA, small molecule inhibitors, and protein inhibitors.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes an inhibitor targeting RYK or an inhibitor targeting G-ECD.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. A combination of biomarkers for detecting the severity of progression of MASLD / MASH and / or pulmonary fibrosis, characterized in that, The biomarker combination includes G-ECD and RYK.

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