CN118001408B - Application of aquaporin 9 phosphorylation inhibitor - Google Patents

Abstract

The present invention discloses a use of a water channel protein 9 phosphorylation inhibitor. The present invention has found through research that hepatocyte water channel protein 9 exists in a phosphorylated state, and inhibiting its phosphorylation has the effect of antagonizing the progression of non-alcoholic fatty liver disease. In addition, the present invention screens the specific kinases for the phosphorylation of water channel protein 9 and develops a water channel protein 9 phosphorylation inhibitor to effectively antagonize the progression of non-alcoholic fatty liver disease.

Description

Use of a water channel protein 9 phosphorylation inhibitor

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to the use of a water channel protein 9 phosphorylation inhibitor, and in particular to the use of the water channel protein 9 phosphorylation inhibitor in the treatment of non-alcoholic fatty liver disease.

Background technique

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world, with a global adult prevalence of approximately 25%. Due to its high prevalence, NAFLD is currently the fastest growing cause of liver-related mortality worldwide, and has become an important cause of primary liver cancer, end-stage liver disease, and liver transplantation, resulting in a huge health and economic burden. Despite increasing attention, its pathogenesis is quite complex and there is a lack of effective treatments. Therefore, further exploring the molecular mechanisms of the occurrence and development of NAFLD and using them to guide the development of new therapeutic drugs and strategies remains a huge challenge.

The occurrence and development of NAFLD is related to the redistribution of triglyceride (TG). Glycerol, as the basic raw material for the synthesis of TG, enters hepatocytes through free diffusion and water glycerol channels. Aquaporin 9 (AQP9) is the main glycerol channel of hepatocytes and can accelerate glycerol uptake. When liver lipid metabolism is disordered, the rapid glycerol uptake function of AQP9 plays an important role. Knocking out Aqp9 can slow the progression of NAFLD induced by a high-fat diet (HFD) and antagonize related metabolic changes. Therefore, AQP9 is an important influencing factor of TG synthesis, and studying its role in hepatocyte lipid metabolism is of great research value. It is not clear whether the post-translational modification of hepatocyte AQP9 affects its function.

Therefore, it is still necessary to study the role of post-translational modification of hepatocyte AQP9 in the pathogenesis of NAFLD to provide a research basis for the development of efficient targeted drugs and prevention strategies.

Summary of the invention

In order to solve at least some of the technical problems in the above-mentioned prior art, the present invention provides the use of aquaporin 9 phosphorylation inhibitor in treating non-alcoholic fatty liver disease. Specifically, the present invention includes the following contents.

In a first aspect of the present invention, there is provided a use of an inhibitor of aquaporin 9 phosphorylation in the preparation of a medicament for preventing, treating or ameliorating non-alcoholic fatty liver disease or conditions related thereto, wherein the inhibitor is capable of directly or indirectly regulating the phosphorylation of aquaporin 9, thereby downregulating or reducing the amount and/or activity of phosphorylated aquaporin 9, or achieving non-phosphorylation of aquaporin 9.

In certain embodiments, according to the use of the present invention, the inhibitor comprises at least one of a small molecule compound, a nucleic acid, a protein, an antibody, an enzyme, a polypeptide and a gene editing tool.

In certain embodiments, according to the use of the present invention, the inhibitor is selected from at least one of the following:

(1) Mutation reagents for non-phosphorylation of aquaporin 9;

(2) inhibitors or antagonists of phosphorylation kinases associated with aquaporin 9;

(3) substances that block the binding of aquaporin 9 to its associated phosphorylation kinase;

(4) An antibody or an antigen-binding fragment thereof directed against phosphorylated aquaporin-9.

In certain embodiments, according to the use described in the present invention, the prevention, treatment or improvement includes at least one of the following: reducing fat droplet accumulation, reducing the degree of liver lobule structural disorder, reducing liver tissue triglyceride content, reducing liver tissue total cholesterol content, reducing serum free fatty acid content, reducing serum total cholesterol content, reducing serum triglyceride content, reducing serum low-density lipoprotein cholesterol content, reducing liver index, reducing liver fibrosis, reducing hepatocyte ballooning, reducing inflammatory response associated with diseased liver tissue, reducing acquired metabolic stress liver damage associated with insulin resistance and genetic susceptibility, and reducing overweight associated with non-alcoholic fatty liver disease and/or metabolic syndrome-related symptoms including visceral obesity, increased fasting blood sugar, dyslipidemia and hypertension.

In certain embodiments, according to the use of the present invention, the phosphorylation of aquaporin-9 includes phosphorylation of serine at position 292 of aquaporin-9.

In certain embodiments, according to the use of the present invention, the inhibitor is KN93.

In certain embodiments, according to the use of the present invention, the inhibitor is a gene drug for mutation of phosphorylation site 9.

The second aspect of the present invention provides the use of aquaporin 9 phosphorylation inhibitors in combination with other therapeutic agents for preventing, treating or improving non-alcoholic fatty liver disease or conditions related thereto.

In a third aspect, the present invention provides an antibody or an antigen-binding fragment thereof against phosphorylated aquaporin-9, which can specifically bind to aquaporin-9 phosphorylated at serine 292.

In certain embodiments, the antibody or antigen-binding fragment thereof against phosphorylated aquaporin 9 according to the present invention comprises a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody.

In a fourth aspect of the present invention, a product for detecting the phosphorylation state of aquaporin 9 is provided, which includes a reagent capable of specifically binding to phosphorylated aquaporin 9, instructions for using the reagent to detect a sample to be tested, and instructions for determining the phosphorylation state of aquaporin 9.

In certain embodiments, the product for detecting the phosphorylation status of aquaporin 9 according to the present invention comprises a kit, a test paper or a chip.

In certain embodiments, in the product for detecting the phosphorylation state of aquaporin 9 according to the present invention, the reagent comprises an antibody.

In certain embodiments, in the product for detecting the phosphorylation state of aquaporin 9 according to the present invention, the antibody comprises a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a mouse antibody.

In a fifth aspect of the present invention, a method for screening a drug for treating or improving non-alcoholic fatty liver disease or a condition related thereto is provided, comprising:

a. Measuring the phosphorylation level of aquaporin 9 in a cell model or an animal model to obtain a first measurement value;

b. contacting the drug to be tested with a cell model or an animal model;

c. measuring the phosphorylation level of aquaporin 9 in the model after contact with the test drug to obtain a second measurement value;

d. When the first measured value is greater than the second measured value, the test substance is screened as a candidate drug for preventing, treating or improving non-alcoholic fatty liver disease or related conditions.

In a sixth aspect of the present invention, a method for regulating the phosphorylation of aquaporin 9 is provided, which comprises the step of treating cells or animals with a reagent, wherein the regulation comprises regulating the phosphorylation of aquaporin 9 directly or indirectly, thereby regulating the amount and/or activity of phosphorylated aquaporin 9, or achieving non-phosphorylation or phosphorylation of aquaporin 9.

In certain embodiments, according to the method for regulating phosphorylation of aquaporin 9 of the present invention, the reagent includes an inhibitor of phosphorylation of aquaporin 9, and preferably a mutant reagent for non-phosphorylation of aquaporin 9, an inhibitor or antagonist of phosphorylation kinase associated with aquaporin 9, a substance that blocks the binding of aquaporin 9 to its related phosphorylation kinase, an antibody against phosphorylated aquaporin 9 or an antigen-binding fragment thereof, so as to achieve downregulation of the amount and/or activity of phosphorylated aquaporin 9, or achieve non-phosphorylation of aquaporin 9.

In certain embodiments, according to the method for regulating phosphorylation of aquaporin 9 of the present invention, the reagent includes an aquaporin 9 phosphorylation activator or promoter, and preferably also includes an activator or promoter of phosphorylation kinase associated with aquaporin 9, a substance that promotes the binding of aquaporin 9 to its associated phosphorylation kinase, and a genetic engineering reagent related to overexpression of phosphorylated aquaporin 9, so as to achieve upregulation of the amount and/or activity of phosphorylated aquaporin 9, or achieve phosphorylation of aquaporin 9, or overexpression of phosphorylated aquaporin 9.

The present invention has found through research that the 292th serine of hepatocyte AQP9 (AQP9 S292) is phosphorylated, and inhibiting the phosphorylation of AQP9 S292 has the effect of antagonizing the progression of NAFLD. In addition, the present invention screened the specific kinase of AQP9 S292 and developed an AQP9 S292 phosphorylation inhibitor to effectively antagonize the progression of NAFLD.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the protein expression level of AQP9 in liver tissue, wherein Figure 1A shows the expression changes of AQP9 in the NAFLD model, two bands of AQP9 were detected at about 37 kDa, the signals of both bands were enhanced, and the enhancement of the large molecular weight band was more obvious; Figure 1B shows the statistical results of the expression changes of AQP9; Figure 1C shows the histochemical staining of the expression changes of AQP9, the liver lobule structure disappeared after modeling, and the expression of AQP9 was significantly increased.

Figure 2 shows the presence of phosphorylated serine residues in AQP9 of mouse liver and Hep G2 cell lines, wherein, in Figure 2 A and B, the Co-IP (co-immunoprecipitation) and reverse Co-IP of AQP9 and phosphorylated serine (pSer) in mouse liver tissue both showed that p-AQP9 in the model group was upregulated; in Figure 2 C, the Co-IP of AQP9-FLAG and pSer in Hep G2 cells showed that p-AQP9 was upregulated after OA treatment.

FIG3 is a screening of liver AQP9 phosphorylation sites, wherein FIG3A screens liver AQP9 phosphorylation sites using mass spectrometry, and finds that AQP9 S292 is phosphorylated; FIG3B shows that AQP9 S292 is highly conserved in mammals such as mice and rats, as well as humans.

FIG4 shows the preparation of polyclonal antibodies against AQP9 S292 phosphorylation, wherein the homemade anti-p-AQP9 S292 antibody can detect AQP9-WT, but has a poor effect in detecting S292 mutants.

FIG5 shows that the phosphorylation of AQP9 S292 is increased in animal models of liver-related metabolic diseases, wherein, in FIG5A , compared with the control group, the phosphorylation of AQP9 S292 and total AQP9 (t-AQP9) in the liver of mice with HFD-induced liver steatosis increased, and the increase of p-AQP9 was more significant than that of t-AQP9; in FIG5B , the phosphorylation levels of AQP9 S292 and t-AQP9 in the liver of CDAHFD-induced obese mice increased, and the increase of p-AQP9 was more significant than that of t-AQP9; in FIG5C , compared with the corresponding age-matched control group, the phosphorylation levels of AQP9 S292 and t-AQP9 in the liver of 8-week-old or 12-week-old male db/db mice increased, and the phosphorylation level of AQP9 S292 increased significantly with the increase of age, while the t-AQP9 in 12-week-old mice decreased compared with the 8-week-old db/db mice.

FIG6 shows the results of transfecting AQP9-WT, AQP-S292A and AQP9-S292D respectively on the basis of knocking down AQP9, among which AQP9-WT and AQP9-S292D both accumulated more lipid droplets, while AQP9-S292A eliminated this phenomenon.

FIG. 7 shows that AQP9 knockout mice are protected against diet-induced lipid accumulation, whereas transfection of the AQP9 S292D mutant, but not AQP9 S292A, into Aqp9 ^−/− mice aggravates NAFLD.

Figure 8 Prediction of phosphorylation kinases of liver AQP9, wherein, in Figure 8A, NetPhos 3.1 predicts the top 6 kinases that may phosphorylate the mouse AQP9 S292 site; in Figure 8B, three independent IP-MS simultaneously identified kinases that interact with AQP9 in Hep G2 cells, CaMKIIδ is one of the 12 kinases identified in the three independent IP-MS experiments; in Figure 8C, CaM is the only calmodulin identified in the three independent IP-MS experiments.

Figure 9 shows that AQP9 interacts with CaMKIIδ, wherein in Figure 9 A, hydrogen bond interactions were determined by Pymol protein-protein interaction analysis, and Phe174 of KCC2D (CaMKIIδ) and Ala118 of AQP9 formed hydrogen bonds. Under the interaction force, KCC2D-AQP9 scored 2419.940, which performed well; Figure 9 B and C show Co-IP and reverse Co-IP when AQP9-FLAG and CaMKIIδ-HA are expressed in Hep G2 cells; Figure 9 D and E show Co-IP and reverse Co-IP of mouse liver AQP9 and CaMKIIδ.

FIG. 10 shows that CaMKIIδ can increase lipid droplet accumulation in AQP9-WT Hep G2 cells, but cannot increase lipid droplet accumulation in AQP9-S292A HepG2 cells.

Figure 11 shows the results of an in vitro kinase experiment of CaMKIIδ and AQP9, wherein AQP9 was phosphorylated by CaMKIIδ in vitro; recombinant AQP9-FLAG was incubated with recombinant CaMKIIδ-HA in vitro; phosphorylation of S292 was detected using a phosphorylation-specific antibody against AQP9 S292; phosphorylation of S292 was detected when AQP9 and CaMKIIδ were present at the same time; however, when KN-93 (CaMKII inhibitor) was added, the phosphorylation level of S292 was reduced, and no phosphorylation was detected in the absence of kinase.

FIG. 12 shows that in Hep G2 cells, overexpression of CaMKIIδ enhanced the phosphorylation of AQP9 S292, while the CaMKII inhibitor KN-93 dose-dependently attenuated the phosphorylation level of S292.

Figure 13 shows the results of HE and ORO staining, in which the lipid droplet accumulation in rats in the CDAHFD+KN93 group was reduced and the liver lobule structure was improved.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

use

The first aspect of the present invention provides the use of a water channel protein 9 phosphorylation inhibitor in the preparation of a medicine for preventing, treating or improving non-alcoholic fatty liver disease or its related conditions. The present invention first discovered and verified through research that phosphorylated water channel protein 9 can be used as a target for inhibiting NAFLD. Therefore, without being bound by any theory, it is possible to regulate water channel protein 9 phosphorylation in a direct or indirect manner, thereby regulating (preferably lowering) the amount and/or activity of phosphorylated water channel protein 9, or any reagent that realizes the non-phosphorylation of water channel protein 9 can be used as an inhibitor of the present invention, for preventing, treating or improving non-alcoholic fatty liver disease or its related conditions. Herein, non-phosphorylation includes the dephosphorylation of phosphorylated water channel protein 9.

Unless otherwise specified, the phosphorylation site referred to in the present invention specifically refers to the serine phosphorylation at position 292 of aquaporin-9.

The present invention also verifies the kinase closely related to phosphorylation through experiments. In a specific embodiment, the kinase interacting with aquaporin 9 is CaMKIIδ.

In the present invention, the inhibitor is selected from at least one of the following: (1) a mutagenic agent for non-phosphorylated aquaporin 9 (examples thereof include but are not limited to chemical mutagens, physical mutagens, biological mutagens, gene editing tools for point mutations, and overexpression genetic engineering agents for non-phosphorylated aquaporin 9, etc.); (2) an inhibitor or antagonist of a phosphorylated kinase associated with aquaporin 9 (examples thereof include but are not limited to antibodies targeting phosphorylated kinases, nucleic acids targeting phosphorylated kinases, gene editing tools for knocking down or knocking down phosphorylated kinases, etc.); (3) a substance that blocks the binding of aquaporin 9 to its associated phosphorylated kinases (examples thereof include but are not limited to antibodies targeting phosphorylated kinases and/or aquaporin 9, or inhibitors of upstream cell signaling pathways involved in the phosphorylation process, or modification of the phosphorylation sites of kinases and/or aquaporin 9 by chemical modification-related reagents, etc.); (4) an antibody or antigen-binding fragment thereof against phosphorylated aquaporin 9.

In some preferred embodiments, the inhibitor includes at least one of a small molecule compound, a nucleic acid, a protein, an antibody, an enzyme, a peptide, and a gene editing tool. Among them, the small molecule compound includes KN93 or other known small molecules equivalent thereto that have the activity of inhibiting phosphorylation kinase (such as KN62, STO609, arcyriaflavin A, Myr-AIP, K252a, K252b, etc.).

In the present invention, examples of nucleic acids that can be used as inhibitors include, but are not limited to, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamers, decoy ODNs, shRNAs, gRNAs, etc., directed against genes encoding phosphorylation kinases. In certain embodiments, the sense and antisense sequences of siRNAs are: 5'-UGAUCGAAGCCAUAAGCAA (dTdT)-3' (SEQ ID NO.1) and 5'-UUGCUUAUGGCUUCGAUCA (dTdT)-3' (SEQ ID NO.2), respectively. In addition to the above sequences, those skilled in the art can also design and synthesize corresponding sequences based on published gene or protein databases, or use any nucleic acid known in the art that can target genes encoding phosphorylation kinases.

In the present invention, examples of polypeptides that can serve as inhibitors include, but are not limited to, autocamtide-2-related inhibitory peptides and the like.

In the present invention, examples of gene editing tools that can be used as inhibitors include, but are not limited to, zinc finger nucleases (ZFNs), transcription activators (TALENs), CRISPR-Cas9 systems, etc. In addition, gene editing reagents or kits for editing the 292 serine site of AQP9 in animal or human cells or tissues also fall within the scope of the present invention. Gene editing tools such as zinc finger editing tools, TALEN editing tools, CRISPR/Cas9 and similar families, single base editing tools, and plasmid vectors and viral infection tools prepared therefrom for the site also fall within the scope of the present invention.

In a preferred embodiment, the inhibitor is KN93.

In a preferred embodiment, the inhibitor is a gene drug, and preferably a genetic engineering agent that promotes overexpression of a non-phosphorylated mutant. In a specific embodiment, the mutant is an AQP9-S292A mutant. It is understood that the gene drug comprises any of the above-mentioned nucleic acid molecules, expression vectors comprising the nucleic acid molecules, and recombinant cells comprising the vectors.

In a preferred embodiment, the nucleotide sequence encoding the AQP9-S292A mutant is shown in SEQ ID NO.3:

ATGCAGCCTGAGGGAGCAGAAAAGGGAAAAAGCTTCAAGCAGAGACTGGTCTTGAAGAGCAGCTTAGCGAAAGAAACCCTCTCTGAGTTCTTGGGCACGTTCATCTTGATTGTCCTTGGATGTGGCTGTGTTGCCCAAGCTATTCTCAGTCGAGGACGTTTTGGAGGGGTCATCACTATCAATGTTGGATTTTCAATGGCAGTTGCAATGGCCATTTATGTGGCTGGCGGTGTCTCTGGTGGTCACATCAACCCAGCTGTGTCTTTAGCAATGTGTCTCTTTGGACGGATGAAATGGTTCAAATTGCCATTTTATGTGGGAGCCCAGTTCTTGGGAGCCTTTGTGGGGGCTGCAACCGTCTTTGGCATTTACTATGATGGACTTATGTCCTTTGCTGGTGGAAAACTGCTGATCGTGGGAGAAAATGCAACAGCACACATTTTT GCAACATACCCAGCTCCGTATCTATCTCTGGCGAACGCATTTGCAGATCAAGTGGTGGCCACCATGATACTCCTCATAATCGTCTTTGCCATCTTTGACTCCAGAAACTTGGGAGCCCCCAGAGGCCTAGAGCCCATTGCCATCGGCCTCCTGATTATTGTCATTGCTTCCTCCCTGGGACTGAACAGTGGCTGTGCCATGAACCCAGCTCGAGACCTGAGTCCCAGACTTTTCACTGCCTTGGCAGGCTGGGGGTTTGAAGTCTTCAGAGCTGGAAACAACTTCTGGTGGATTCCTGTAGTGGGCCCTTTGGTTGGTGCTGTCATTGGAGGCCTCATCTATGTTCTTGTCATTGAAATCCACCATCCAGAGCCTGACTCAGTCTTTAAGACAGAACAATCTGAGGACAAACCAGAGAAATATGAACTCGCTGTCATCATGTAG.

In the present invention, the term "treatment" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) the progression of undesirable physiological changes or disorders, such as non-alcoholic fatty liver disease. Compared with an untreated reference group under the same conditions, the degree of relief or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique. Beneficial or desired clinical results include, but are not limited to, the following detectable or undetectable results, including relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete). "Treatment" also refers to the extended survival period compared to the expected survival period when not receiving treatment. Those in need of treatment include those who already have non-alcoholic fatty liver disease or related conditions or those who need to prevent or improve non-alcoholic fatty liver disease or related conditions.

Combined

The second aspect of the present invention provides the use of aquaporin 9 phosphorylation inhibitors in combination with other therapeutic agents in the preparation of a medicament for preventing, treating or ameliorating non-alcoholic fatty liver disease or conditions related thereto.

In the present invention, other therapeutic agents include but are not limited to vitamins, lipid-lowering drugs, insulin sensitizing drugs, anti-inflammatory drugs, cholesterol-lowering drugs, diabetes drugs, experimental NASH inhibitors and weight-loss drugs. Among them, examples of the vitamins include but are not limited to vitamin B, vitamin D or vitamin E. Examples of the lipid-lowering drugs include but are not limited to liraglutide, exenatide, albiglutide, acipimox, atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, pravastatin, ezetimibe, etc. Examples of the anti-inflammatory drugs include but are not limited to antioxidant drugs (such as resveratrol, caffeic acid, dihydromyricetin, soybean lecithin, etc.), anti-apoptotic drugs (such as growth factors, antioxidants, Caspase family inhibitors, Bcl 2 family, PARP inhibitors, Calpain blockers, etc.) and anti-cytokine drugs (such as IFN-α, IFN-β, IFN-γ, IFN-ε, GM-CSF, G-CSF) at least one. Examples of the diabetes drugs include, but are not limited to, oral hypoglycemic drugs and injection preparations, wherein examples of oral hypoglycemic drugs include, but are not limited to, sulfonylureas (e.g., gliclazide, glimepiride), glinides (e.g., repaglinide), biguanides (e.g., metformin), thiazolidinediones, α-glucosidase inhibitors (e.g., acarbose), DDP-4 inhibitors, and SGLT-2 inhibitors, etc.; examples of injection preparations include, but are not limited to, insulin, insulin analogs, and GLP-1 receptor agonists, etc. The experimental NASH inhibitor is selected from at least one of farnesoid x receptor agonists, PPAR agonists, acetyl-CoA carboxylase (ACC), C-C chemokine ligand type 2 and type 5 antagonists, apoptosis signal regulating kinase (ASK1) inhibitors, lysyl oxidase-like 2 antibodies, anti-liver fibrosis agents, and galectin-3 inhibitors. Examples of anti-obesity drugs include, but are not limited to, peptide YY or its analogs, neuropeptide Y receptor type 2 (NPYR2) agonists, NPYR1 or NPYR5 antagonists, lipase inhibitors (e.g., orlistat), human pro-islet peptide (HIP), melanocortin receptor 4 agonists (e.g., semaphoride), melanin-aggregating hormone receptor 1 antagonists, farnesoid X receptor (FXR) agonists (e.g., obeticholic acid), zonisamide, phentermine (alone or in combination with topiramate), norepinephrine/dopamine reuptake inhibitors (e.g., bupropion), norepinephrine/dopamine reuptake inhibitors, GDF-15 analogs, cholecystokinin agonists, amylin and its analogs (e.g., pramlintide), leptin and its analogs (e.g., metreleptin), serotonin activators (e.g., lorcaserin), methionine aminopeptidase 2 (MetAP2) inhibitors (e.g., beloranib or ZGN-1061), phendimetrazine, amfepramone, benzphetamine, SGLT2 inhibitors (e.g., empagliflozin, canagliflozin, dapagliflozin, empagliflozin, togliflozin, sergliflozin etabonate, repagliflozin etabonate, or empagliflozin), SGLTL1 inhibitors, dual SGLT2/SGLT1 inhibitors, fibroblast growth factor receptor (FGFR) modulators, AMP-activated protein kinase (AMPK) activators, biotin, MAS receptor modulators, or glucagon receptor agonists (alone or in combination with another GLP-1R agonist (e.g., liraglutide, exenatide, dulaglutide, albiglutide, lisenatide, or shimaglutide)).

Aquaporin 9 phosphorylation detection antibody and detection method

The third aspect of the present invention provides an antibody or an antigen-binding fragment thereof against phosphorylated aquaporin 9, which can specifically bind to aquaporin 9 phosphorylated at serine 292, so that it can be used as a detection antibody for detecting the level of phosphorylated aquaporin 9.

In the present invention, the antibody includes monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody or mouse antibody.

In the present invention, the antigen-binding fragment includes at least one of Fab, Fab', F(ab)2, F(ab')2, scFv, scFv Fc fragment or single-chain antibody ScAb.

The present invention prepares a detection antibody that can specifically bind to aquaporin 9 phosphorylated at serine 292. The preparation method is not particularly limited, including preparation by artificial synthesis or genetic engineering. In certain embodiments, the antibody of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibody of the present invention or its antigen-binding fragment is obtained by direct amino acid synthesis. In certain embodiments, recombinant technology can be used to obtain the antibody in large quantities. An exemplary method is to clone its encoding gene into a vector, then transfer it into a cell, and then separate it from the host cell after proliferation by conventional methods.

The fourth aspect of the present invention provides a product for detecting the phosphorylation state of aquaporin 9, which includes a reagent that can specifically bind to phosphorylated aquaporin 9, and instructions for using the reagent to detect a sample to be tested and instructions for determining the phosphorylation state of aquaporin 9.

In the present invention, the detection product includes but is not limited to a test kit, a test paper or a chip.

In a preferred embodiment, the reagent includes the above-mentioned detection antibody.

Drug Screening

In a fifth aspect of the present invention, there is provided a method for screening a drug for treating or improving non-alcoholic fatty liver disease or a condition related thereto, comprising:

a. Measuring the phosphorylation level (amount and/or activity) of aquaporin 9 in a cell model or an animal model to obtain a first measurement value;

b. contacting the drug to be tested with a cell model or an animal model;

c. measuring the phosphorylation level of aquaporin 9 in the model after contact with the test drug to obtain a second measurement value;

d. When the first measured value is greater than the second measured value, the drug to be tested is screened as a candidate drug for preventing, treating or improving non-alcoholic fatty liver disease or related conditions.

In a preferred embodiment, the cells are cells derived from the Hep G2 cell line.

In another preferred embodiment, the animal model is a mouse.

Methods for regulating phosphorylation of aquaporin 9

A sixth aspect of the present invention provides a method for regulating phosphorylation of aquaporin 9. In one embodiment, the method is an in vitro method. In another embodiment, the method is a non-therapeutic or diagnostic method, such as for research or drug optimization (including drug design and structure optimization, drug screening, etc.).

In a preferred embodiment, the method comprises the step of treating cells or animals with an agent, wherein the regulation comprises regulating the phosphorylation of aquaporin 9 directly or indirectly, thereby regulating the amount and/or activity of phosphorylated aquaporin 9, or achieving non-phosphorylation or phosphorylation of aquaporin 9.

In a preferred embodiment, the reagent includes an aquaporin 9 phosphorylation activator or promoter, and preferably includes an activator or promoter of a phosphorylation kinase associated with aquaporin 9, a substance that promotes the binding of aquaporin 9 to its associated phosphorylation kinase, and a genetic engineering reagent associated with the overexpression of phosphorylated aquaporin 9, so as to achieve the upregulation of the amount and/or activity of phosphorylated aquaporin 9, or to achieve the phosphorylation of aquaporin 9, or the overexpression of phosphorylated aquaporin 9. The genetic engineering reagent associated with the overexpression of phosphorylated aquaporin 9 includes a nucleic acid molecule capable of expressing a mutant protein with a mutation at the 292nd serine site of phosphorylated aquaporin 9, an expression vector containing the nucleic acid molecule, and at least one of a host cell containing the mutant protein, the nucleic acid molecule, and the expression vector. For example, by using gene editing technology, a site-directed mutation is performed on the coding sequence corresponding to the 292th serine site on the AQP9 gene in the cell, or a mutant protein with serine phosphorylation at 292 of AQP9 is overexpressed in the cell using gene overexpression technology. In a specific embodiment, the mutant protein is AQP9-S292D.

In another preferred embodiment, the reagent includes an inhibitor of phosphorylation of aquaporin 9, and preferably a mutant reagent for non-phosphorylation of aquaporin 9, an inhibitor or antagonist of phosphorylation kinase associated with aquaporin 9, a substance that blocks the binding of aquaporin 9 to its associated phosphorylation kinase, an antibody or antigen-binding fragment thereof against phosphorylated aquaporin 9, and a genetic engineering reagent associated with dephosphorylation of aquaporin 9, so as to achieve down-regulation of the amount and/or activity of phosphorylated aquaporin 9, or to achieve non-phosphorylation of aquaporin 9. The genetic engineering reagent associated with non-phosphorylation of aquaporin 9 includes a nucleic acid molecule capable of expressing a mutant protein of a dephosphorylation mutation of serine 292 of aquaporin 9, an expression vector containing the nucleic acid molecule (including but not limited to a plasmid vector, a phage vector and a viral vector, such as a lentiviral vector and an adeno-associated viral vector), and a host cell containing the mutant protein, the nucleic acid molecule, and the expression vector. For example, by using gene editing technology, the coding sequence corresponding to the serine site 292 on the AQP9 gene is subjected to site-directed mutation in the cell, or by using gene overexpression technology, a mutant protein is produced by dephosphorylating the serine 292 of AQP9 in the cell. In a specific embodiment, the mutant protein is AQP9-S292A.

Example 1

This example shows the analysis of AQP9 phosphorylation sites in hepatocytes.

In order to analyze whether AQP9 in hepatocytes is phosphorylated, two similar AQP9 bands were detected at about 37 kDa by Western Blot experiment (Figure 1A). The expression level in the model group increased, and the high molecular weight band was more obvious (Figure 1B). In order to determine whether phosphorylated AQP9 is one of the two bands, liver tissue samples were used to immunoprecipitate AQP9 and immunoblot for phosphorylated serine residues. AQP9 serine phosphorylation was detected and increased in the model group (Figure 2A). Immunoprecipitation of phosphorylated serine residues and immunoblotting of AQP9 in liver tissues also increased AQP9 phosphorylation in the model group (Figure 2B). Then AQP9-FLAG was transfected into Hep G2 cells, and FLAG immunoprecipitation and immunoblotting of phosphorylated serine residues were performed. The results showed that the phosphorylation level of AQP9-FLAG increased after oleic acid treatment (Figure 2C). This result suggests that AQP9 contains phosphorylatable serine residues and that phosphorylation is increased under CDAHFD or OA treatment.

To explore the specific AQP9 phosphorylation site, phosphorylation mass spectrometry analysis was applied, and only one potential phosphorylated serine residue, S292, was identified in AQP9 in mouse liver tissue (Figure 3A). S292 is conserved in several mammalian species (Figure 3B), indicating that it is essential for the biological function of AQP9.

Example 2

This example shows the analysis of the detection effect of AQP9 S292 phosphorylation antibody.

In order to characterize the phosphorylation of AQP9 S292 site and evaluate its potential physiological and pathological significance, the present invention developed a customized polyclonal antibody (anti-p-AQP9 S292) that specifically reacts with phosphorylated AQP9 S292. In Hep G2 cells expressing FLAG-tagged wild-type AQP9, S292A mutant or S292D mutant, the prepared antibody can detect obvious AQP9-WT, while the detection bands of AQP9-S292A or AQP9-S292D are very weak (Figure 4), which meets the validation criteria of phosphorylation antibodies.

Example 3

This example shows the analysis of AQP9 S292 phosphorylation levels.

To detect changes in the phosphorylation level of AQP9 S292 in metabolic-related chronic liver diseases, anti-p-AQP9S292 antibodies were used to first evaluate the phosphorylation status of AQP9 in the liver of diet-induced NAFLD mice, including HFD and CDAHFD-induced models. Compared with the control group, the AQP9 protein level in the HFD or CDAHFD group was significantly increased (A and B in Figure 5), and more importantly, the increase in phosphorylated AQP9 S292 (p-AQP9) was more obvious, resulting in an increase in the ratio of p-AQP9 to total AQP9 (t-AQP9). The present invention also evaluated the phosphorylation of AQP9 at the S292 site in the liver of diabetic mice. Compared with control mice, the S292 phosphorylation level of 8-week-old db/db mice was higher (C in Figure 5). With the increase in insulin levels in the blood, the phosphorylation increase in 12-week-old db/db mice was more obvious, but the t-AQP9 protein did not increase significantly. These results suggest that AQP9 S292 phosphorylation is elevated in states of metabolic dysfunction such as NAFLD or diabetes.

Example 4

This example shows that blocking AQP9 S292 can antagonize the progression of NAFLD.

In order to explore the biological function of AQP9 S292 in NAFLD, the present invention uses shRNA targeting Aqp9 to knock down Aqp9 in HepG2 cells, and then introduces AQP9-WT, AQP9-S292A and AQP9-S292D respectively. In cells overexpressing AQP9-WT or AQP9-S292D, lipid droplets accumulate more, while cells transfected with AQP9-S292A have fewer lipid droplets (Figure 6). It shows that S292A significantly weakens the glycerol uptake capacity of AQP9. The biological function of AQP9 S292 in the NAFLD mouse model was further studied. AQP9-S292A and AQP9-S292D were overexpressed in Aqp9 ^-/- mice. On this basis, CDAHFD was fed. Morphologically, Aqp9 ^-/- mice had fewer lipid droplets in the liver, and Aqp9-S292D-overexpressing Aqp9 ^-/- mice had more lipid droplets in the liver, however, no similar phenomenon was observed in S292A-overexpressing Aqp9 ^-/- mice (Figure 7). Taken together, these data suggest that dephosphorylation of AQP9 at the S292 site can antagonize the progression of NAFLD.

Example 5

This example demonstrates the screening of candidate kinases for hepatocyte AQP9 S292.

In order to screen the kinases that phosphorylate AQP9 S292, the present invention uses NetPhos3.1 to predict its potential kinases based on the peptide sequence near S292. PKA, GSK3, CaMKII, cdc2, CKII and CKⅠ were identified as candidate kinases (Figure 8A). The present invention also immunoprecipitated AQP9 from AQP9-FLAG transfected Hep G2 cells, and then performed mass spectrometry analysis on the immune complex to determine the kinases that may interact. Consistent with the predicted results, CaMKIIδ was one of the 12 kinases identified in three independent IP-MS experiments (Figure 8B). CaM was the only calmodulin identified in three independent IP-MS experiments (Figure 8C).

Example 6

This example shows the analysis of the interaction between AQP9 and CaMKIIδ.

In order to analyze the interaction between AQP9 and CaMKⅡδ, the present invention used PyMOL protein-protein interaction analysis and found that Phe174 of CaMKⅡδ formed a hydrogen bond with Ala118 of AQP9, and the two were well bound (Figure 9 A). Co-IP and reverse Co-IP of AQP9-FLAG and CaMKⅡδ-HA co-expressed in Hep G2 cells showed that AQP9 and CaMKIIδ were located in the same complex (Figure 9 B and C), and Co-IP and reverse Co-IP of endogenous AQP9 and CaMKIIδ in mouse liver tissue further confirmed their interaction (Figure 9 D and E).

Example 7

This example shows the phosphorylation of AQP9 S292 by CaMKIIδ.

In order to study whether CaMKⅡδ phosphorylates AQP9 S292, the present invention uses Hep G2 cell line as a carrier. When CaMKIIδ is present, the glycerol uptake ability of AQP9-WT is significantly enhanced, resulting in increased lipid droplet synthesis (Figure 10). Since AQP9-S292A cannot be phosphorylated by CaMKIIδ, the glycerol uptake ability of AQP9-S292A is not affected (Figure 10). In an in vitro kinase experiment, the purified AQP9-c-Myc protein was incubated with ATP in the presence or absence of CaMKIIδ-c-Myc protein. When AQP9 and CaMKIIδ are present at the same time, AQP9 is phosphorylated (Figure 11). After adding the inhibitor KN93 of CaMKII, the signal of p-AQP9 S292 is weakened (Figure 11). These results strongly indicate that CaMKIIδ is a kinase that phosphorylates AQP9 S292. Using Hep G2 cells as carriers and anti-p-AQP9 S292 antibody, it was found that transfection of CaMKIIδ significantly enhanced the phosphorylation level of AQP9S292 (Figure 12), while KN93 inhibited CaMKIIδ-mediated S292 phosphorylation in a dose-dependent manner.

Example 8

This example shows the therapeutic effect of CaMKII inhibitor KN93 on NAFLD.

In order to clarify the effect of KN93 on NAFLD, the present invention adopts a NAFLD model and intraperitoneally injects KN93. Intraperitoneal injection of KN93 can significantly reduce diet-induced hepatic steatosis and slow down the progression of NAFLD (Figure 13).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (2)

1. A method for screening a drug for treating or improving non-alcoholic fatty liver disease, comprising: a. respectively measuring the phosphorylation level of aquaporin 9 and glycerol uptake of a cell model derived from a Hep G2 cell line in which Aqp9 is knocked down, and obtaining the first measured values of the phosphorylation level of aquaporin 9 and glycerol uptake; b. contacting the test drug with the cell model; c. respectively measuring the phosphorylation level of aquaporin 9 and glycerol uptake in the cell model after contact with the test drug to obtain a second measurement value of the phosphorylation level of aquaporin 9 and glycerol uptake; d. when the first measured value is greater than the second measured value, screening the drug to be tested as a candidate drug for treating or improving non-alcoholic fatty liver disease; or a'. Measuring the phosphorylation level of aquaporin 9 and glycerol uptake in the NAFLD animal model to obtain the first measured value of the phosphorylation level of aquaporin 9 and glycerol uptake; b'. contacting the test drug with the animal model; c 'respectively measuring the phosphorylation level of aquaporin 9 and glycerol uptake in the animal model after contact with the test drug to obtain a second measurement of the phosphorylation level of aquaporin 9 and glycerol uptake; d'. When the first measured value is greater than the second measured value, the drug to be tested is screened as a candidate drug for treating or improving non-alcoholic fatty liver disease; The phosphorylation of aquaporin 9 is the phosphorylation of serine at position 292 of aquaporin 9. 2. The use of KN93 in the preparation of a reagent for regulating the phosphorylation of aquaporin 9 in vitro, characterized in that the use includes the step of treating a cell model with the reagent, wherein the KN93 inhibits CaMKIIδ-mediated serine phosphorylation at position 292 of aquaporin 9 in a dose-dependent manner.